ABSTRACT The functional study of lncRNAs in skeletal muscle satellite cells (SCs) remains at the infancy stage. Here we identify _SAM_ (Sugt1 asssociated muscle) lncRNA that is enriched in

the proliferating myoblasts. Global deletion of _SAM_ has no overt effect on mice but impairs adult muscle regeneration following acute damage; it also exacerbates the chronic injury-induced

dystrophic phenotype in mdx mice. Consistently, inducible deletion of _SAM_ in SCs leads to deficiency in muscle regeneration. Further examination reveals that _SAM_ loss results in a 

cell-autonomous defect in the proliferative expansion of myoblasts. Mechanistically, we find _SAM_ interacts and stabilizes Sugt1, a co-chaperon protein key to kinetochore assembly during

BINDING TO 5′UTR G-QUADRUPLEX IS ESSENTIAL FOR MUSCLE STEM-CELL REGENERATIVE FUNCTIONS Article Open access 19 August 2021 DEPLETION OF SAM LEADING TO LOSS OF HETEROCHROMATIN DRIVES MUSCLE

STEM CELL AGEING Article 19 January 2024 LNCRNA-FKBP1C REGULATES MUSCLE FIBER TYPE SWITCHING BY AFFECTING THE STABILITY OF MYH1B Article Open access 09 April 2021 INTRODUCTION Skeletal

muscle has a robust regenerative capacity, which mainly relies on the activation of resident muscle stem cells, termed satellite cells (SCs). These cells are uniquely marked by the

expression of paired box 7 (Pax7) protein and normally lie in a niche beneath the basal lamina of myofibers in a quiescent stage. Upon injury, they are rapidly activated to enter the cell

cycle and undergo proliferative expansion as myoblast (MB) cells which then differentiate and fuse to form multinucleated myotube (MT) cells; these myotubes further mature into myofibers to

restore the damaged muscle. Meanwhile, a subset of SCs exit the cell cycle and return to the quiescent stage for replenishing the adult stem cell pool. Fine-tuned regulation of cell cycle is

thus essential to ensure appropriate progression through the various overlapping states: activation, proliferation, differentiation, and self-renewal/returning to quiescence. The cell cycle

involves DNA replication and subsequent chromosome separation. The faithful chromosome segregation relies on the assembly of mitotic kinetochore on centromeric chromatin to mediate its

interaction with spindle microtubules1. In vertebrates, the kinetochore is a multilayered disc structure that contains more than a hundred of proteins components2. CCAN, the constitutive

centromere-associated network, is restricted to the centromeres throughout the cell cycle forming a major component of inner kinetochore whereas KMN network, including the KNL1 complex

(containing Knl1 (kinetochore scaffold 1), and Zwint (ZW10 interactor)), the MIS12 complex (containing Mis12, Dsn1, Pmf1 (polyamine-modulated factor 1) and Nsl1), and the NDC80 complex

(containing Ndc80 (also called Hec1), Nuf2, Spc24, and Spc25), is recruited to the centromere by the CCAN during specific stages of mitosis, forming prominent subunits of outer

kinetochore3,4. Among the KMN complexes, Mis12 complex is the keystone to serve as a protein interaction hub which assembles outer kinetochore and links to the inner kinetochore5. Ndc80

complex directly interacts with microtubules through its component Hec16. Given the large number of kinetochore components, its proper assembly is a dynamic and highly orchestrated process.

Any error in kinetochore assembly such as improper targeting or turnover of any component can affect the progression of mitosis, leading to disrupted microtubule attachment, improper

chromosomal segregation, the formation of multipolar spindles, mitotic delay or aneuploidy, etc.7,8,9. It is thus important to elucidate the regulatory mechanisms facilitating kinetochore

assembly, which has not been done in SCs. It is known that SGT1, suppressor of G2 allele of SKP1 (_S. cerevisiae_) (Sugt1) is a highly conserved protein involved in kinetochore assembly10.

As a co-chaperone for Hsp90 protein, mammalian Sugt1 ensures efficient formation of microtubule-binding sites by recruiting Mis12 complexes to kinetochore11. Reduction of Sugt1 in Hela cells

leads to destabilization and mis-localization of Dsn1 and Hec1, thus causing inefficient formation of high-affinity kinetochore-microtubule attachment sites and a mitotic delay10,11. A

recent study also showed that a regulatory phosphatase PHLPP1 dephosphorylates Sugt1 thereby prevents Sugt1 from associating with E3 ligase in turn, countering Sugt1 ubiquitination and

degradation during kinetochore formation12. Long non-coding RNAs (lncRNAs) are emerging as a family of gene regulators of skeletal muscle regeneration and SC activities. Thousands of lncRNAs

have been identified in skeletal muscle cells but our understanding of lncRNA participation in skeletal myogenesis is still at the infancy stage with only a handful of reports from our

group and others13,14,15,16,17,18. Most efforts concentrated on illuminating their regulatory mechanisms in the transition of MB into MT using a mouse MB line, C2C1213,14,15; it remains

largely uncharacterized whether lncRNAs can regulate other states of SCs. In terms of underpinning molecular mechanisms, lncRNAs are best known for engaging in transcriptional and epigenetic

regulation on chromatins, usually through their interaction with chromatin regulators19; other unique mechanisms are also being uncovered to explain the diversified modes of lncRNA actions.

For example, recently, lncRNAs generated from the repeat region of centromere in Drosophila and human, were found to bind to the kinetochore component CENP-C, adding lncRNA to the complex

epigenetic marks at centromeres20,21. Still, it is not known whether non-centromeric lncRNAs exist to interact with proteins involved in kinetochore assembly. Additionally, in vivo

functional analysis is in general lacking for most lncRNAs studied so far despite a wealth of knowledge accumulated from using in vitro cell culture; to date there have been only a few

lncRNA genetically studied using knockout (KO) animals22,23. Here, we have identified one lncRNA, _SAM_, as a regulator of MB proliferation. Its expression is evidently upregulated when SCs

undergo active proliferation; knockdown of _SAM_ in vitro delays proliferative expansion of cells. To further investigate its function in vivo, we generated a KO mouse of _SAM_ using

KO-first strategy; loss of _SAM_ does not cause overt phenotype but indeed leads to impaired regeneration after acute injury. Consistently, inducible deletion of _SAM_ in SCs also delays the

process of acute injury-induced muscle regeneration. Moreover, deletion of _SAM_ in a dystrophic mdx mouse exacerbates the chronic injury-induced dystrophic phenotype. Further examination

reveals that _SAM_ deletion results in the cell-autonomous defect in MB proliferation, pointing to _SAM_ as a promoting factor of MB proliferation. High throughput identification of _SAM_

interacting protein partners reveals that it can specifically bind to Sugt1 and stabilizes its protein level in MBs; loss of _SAM_ causes increased ubiquitination of Sugt1. Mechanistically,

_SAM_ facilitates Sugt1-mediated kinetochore assembly. Loss of _SAM_ or Sugt1 both causes disrupted chromosome alignment and microtubule attachment, which is likely a result of

mis-localization of Dsn1 and Hec1 proteins in centromere. Altogether our findings have identified _SAM_ as a regulator of MB proliferation through its synergistic action with Sugt1 to

promote kinetochore assembly during cell division. RESULTS _SAM_ IS ENRICHED IN MB AND PROMOTES CELL PROLIFERATION Previously we have defined dozens of uncharacterized lncRNAs from C2C12 MB

vs. MT cells through de novo discovery approach integrating RNA-seq and ChIP-seq datasets13. One lncRNA, _Gm11974_, named as Sugt1 Associated Muscle (_SAM_) lncRNA in the present study,

displayed relatively high expression and unexplored function in MB cells (Fig. 1a). It localizes on mouse chromosome 11, in the intervening region of _Myo1g_ (Myosin IG) and _Ccm2_ (Cerebral

cavernous malformation 2) protein-coding genes (Fig. 1b), with well-defined gene structure and a binding peak of myogenic master transcription factor, MyoD on its promoter region (Fig. 1a).

A human homolog of this gene, _SNHG15_, has been studied in cancer, showing upregulated expression in multiple tumor tissues or cells24,25,26 and it promotes cancer cell proliferation and

migration by serving as a sponge for miRNAs27,28,29. Through rapid amplification with cDNA ends (RACE), one dominant isoform was cloned from C2C12 MB cells, which was 592 bp long with four

exons (Fig. 1c). It was predicted as a non-coding RNA by iSeeRNA30 (Supplementary Fig. 1a), consistent with its annotation in the RefSeq (Accession no. NR 045893). _SAM_ was readily detected

in C2C12 MBs and downregulated when the cells underwent differentiation to form MTs (Supplementary Fig. 1b). Consistently, it was enriched in the primary MBs isolated from the skeletal

muscle compared with the whole muscle tissue (Fig. 1d). To further examine its expression dynamics during SC lineage progression, freshly isolated SCs (FISCs) from limb muscles of Pax7-nGFP

mice31 were cultured with growth medium to become activated (ASCs or MBs) which were further cultured to differentiate (DSCs); _SAM_ level was evidently induced (4.7 fold) in ASCs vs. FISCs

but decreased sharply (72.71%) in DSCs vs. FISCs (Fig. 1e). Interestingly, _SAM_ expression appeared not to be heterogeneous in SCs, since no significant difference was detected in the

isolated Pax7High and Pax7Low subpopulations32 of FISCs or ASCs (Supplementary Fig. 1c, d). The above results suggested that _SAM_ might promote MB proliferation. RNA fluorescence in situ

hybridization (RNA-FISH) analysis revealed that _SAM_ transcripts mainly distributed in the cytoplasm of SC (Fig. 1f); a stronger signal was detected in ASC vs. FISC or DSC. Similarly, the

predominant cytoplasmic localization was also observed in C2C12 MB but decreased in MT (Supplementary Fig. 1e). Consistently, cellular fractionation assay in ASCs (Fig. 1g) or C2C12

(Supplementary Fig. 1f) also showed that _SAM_ transcripts were enriched in cytoplasmic extracts, in a similar pattern as _Gapdh_ transcripts, whereas lncRNA _Malat1_ was only found in

nuclear extracts16. The unique cytoplasmic localization of _SAM_ suggested that its function may be distinct from many lncRNAs that are involved in transcriptional regulation in

myogenesis17, which therefore triggered our further investigation. To test if _SAM_ is required for efficient MB proliferation, we knocked down _SAM_ expression in ASCs with two different

siRNA oligos (22.98% and 33.27% reduction, respectively) (Fig. 1h). Forty-eight-hour post-transfection, SCs were stained for Pax7 and MyoD to evaluate the degree of proliferation. It is

known that fully activated SCs are marked by both Pax7 and MyoD while self-renewing SCs express Pax7, but not MyoD; In DSCs, Pax7 expression is lost while Myogenin (MyoG) expression

increases33. Indeed, the percentage of Pax7+MyoD+ cells was markedly reduced upon si_SAM_ knockdown (14.43% and 29.85%, respectively) (Fig. 1i, Supplementary Fig. 1g and h). This was further

confirmed by staining for Ki67; the percentage of Ki67+ SCs was decreased upon _SAM_ loss (48.11% and 55.25%) (Fig. 1j). On the contrary, when overexpressing _SAM_ by transfecting a

_SAM_-expressing plasmid (Fig. 1k), an increase in the percentage of Pax7+MyoD+ (7.16%) or Ki67+ (74.89%) cells was observed (Fig. 1l, m, Supplementary Fig. 1i and j). Altogether, the above

results from loss and gain-of-function assays in vitro on SCs demonstrated that _SAM_ promotes MB proliferation. When performing similar assays using C2C12 MB cell line with stable _SAM_

knockdown by a shRNA, the same overall conclusions were reached (Supplementary Fig. 1k–p). In addition, by cell cycle analysis of synchronized cells, sh_SAM_ cells displayed a higher

percentage of cells in G1 phase at both 12 h and 24 h compared with control (Ctrl) cells, suggesting _SAM_ loss caused cell cycle arrest at G1 phase (Supplementary Fig. 1q); nevertheless,

_SAM_ expression did not show dynamic pattern during the cell cycle progression (Supplementary Fig. 1r). Next, we also examined whether the loss of _SAM_ has any effect on MB

differentiation. Single myofibers were isolated from extensor digitorum longus (EDL) muscle of mouse and transfected with si_SAM_. Staining with MyoG and MyoD 72 h post-transfection revealed

that the percentage of MyoD+MyoG+ cells was significantly increased (16.43% and 19.53%) upon _SAM_ knockdown (Fig. 1n, Supplementary Fig. 1s and t), implying that these cells may have

precocious differentiation potential. Collectively, these findings from the in vitro cultured cells indicate that _SAM_ is necessary for maintaining proper myogenic proliferation and

preventing precocious differentiation. _SAM_ DELETION IN MOUSE IMPAIRS MUSCLE REGENERATION To further elucidate the functional roles of _SAM_ in vivo, we generated a KO mouse. Given that

lncRNA locus may function as an enhancer region to regulate gene expression34 and active enhancer mark, H3K27ac, was indeed found on _SAM_ locus (Fig. 1a), we thus employed a KO-first

strategy that ablates gene function by inserting RNA processing signals without deletion of the target locus. As illustrated in Fig. 2a, the KO-first allele was generated by inserting a

splicer acceptor (SA)-internal ribosomal entry site (IRES)-LacZ cassette and a Neo-polyadenylation (pA) signal into the intron 2, thus achieving the disruption of _SAM_ transcription. The

insertion was flanked by FRT sites that will allow Flippase recombinase to remove the gene-trapping cassette, hereby converting the KO to a conditional allele with loxP sites flanking exons

3–4. DNA genotyping confirmed the insertion of the SA-IRES-LacZ-pA cassette in the KO mouse genome (Supplementary Fig. 2a, b). Three qRT-PCR primers targeting different regions (exons 1–2,

exons 2–3, and exons 3–4) were used to detect possible transcription (Fig. 2a); and no transcripts were detected with any pair of primers in the isolated SCs (Fig. 2b) or tested tissues

(Supplementary Fig. 2c). It is interesting that no truncated transcript from exons 1–2 was detected despite the PolyA was inserted after exon 2. To test if non-sense-mediated RNA decay had

possibly led to degradation of the transcript, we found treatment with cycloheximide (CHX), which is known to reverse non-sense-mediated RNA decay35 induced the appearance of the truncated

transcript from exons 1–2 (Supplementary Fig. 2d). Examining the adult mouse phenotype, we found that the KO mice were viable, fertile without overt morphological deformities (Fig. 2c);

consistently, the size and weight of the KO mice were comparable with the WT littermates (Fig. 2c, d). Histological analyses of adult tissues including liver, spleen, lung, kidney, and ovary

also revealed no overt differences between the KO and WT littermates (Supplementary Fig. 2e); similarly, when examining the adult skeletal muscle at 8 weeks, the fibers also appeared normal

in size and pattern (Fig. 2e), showing the deletion of _SAM_ may not have any impact on the adult muscle development. In addition, the number of Pax7+ quiescent SCs (QSCs) was not changed

in the muscles of the KO mice (Fig. 2f), indicating that _SAM_ may not be required for maintenance of the SC pool. Lastly, examining muscle formation at embryonic (E18.5) (Supplementary Fig.

 2f, g) or postnatal (P7) days (Supplementary Fig. 2h) revealed no overt changes in muscle morphology and the number of Pax7+ cells in WT vs. KO mice, suggesting that _SAM_ may not play a

role in embryonic or postnatal myogenesis. Considering the promoting function of _SAM_ that was uncovered above in proliferating MB in vitro (Fig. 1), we speculated that loss of _SAM_ may

have an impact on muscle regeneration in vivo. To test this notion, BaCl2 was injected into the tibialis anterior (TA) muscles of 8–9 weeks old mice to induce massive myofiber necrosis

followed by immune cell infiltration, activation, and proliferation of SCs, which then formed new myofibers to repair the damaged fibers within 3–4 weeks post injection. The newly formed

myofibers were normally characterized by centrally localized nuclei (CLN) and expression of embryonic MyHC (eMyHC) protein. The above injected muscles were collected 4, 7, 14, and 28 days

after the injury for evaluation of the degree of muscle regeneration (Fig. 2g). Indeed, by H&E staining, the number of CLN+ fibers per field was evidently decreased (32.29%) in KO vs. WT

mice 4 days after the injury (Fig. 2h); consistently, the number of eMyHC+ fibers was also decreased by 20.16% (Fig. 2i). Nevertheless, by day 7, no significant difference was found in KO

vs. WT mice; by day 28, the damaged muscle fibers were fully regenerated in both mice (Fig. 2h, i). The above results indicate that _SAM_ deletion causes a delay but not a blockage in

injury-induced muscle regeneration. In addition, we found that Pax7+ or MyoD+ cells were both reduced significantly (39.44% and 38.81%, respectively) on the KO muscles compared with WT (Fig.

 2j, k) 3 days after injury, suggesting a decline in the expansion of SC progeny during the regeneration process. Lastly, we quantified the number of Pax7+ cells one month after injury when

SCs were expected to return to quiescence (Supplementary Fig. 2i); no significant difference was observed from the injured muscles of KO vs. WT mice (Supplementary Fig. 2j), suggesting _SAM_

ablation may not exert apparent influence on SC self-renewal during muscle regeneration. INDUCIBLE ABLATION OF _SAM_ IN SC DELAYS MUSCLE REGENERATION To further pinpoint that the above

described regeneration phenotype is attributed to the loss of _SAM_ in SCs, we further generated a SC-specific inducible knockout (iKO) mouse. As illustrated in Fig. 2l, _SAM_ floxed mice

(_SAM_fl/fl) were created by crossing the KO with a FLPeR recombinase mouse, which led to the excision of the SA-IRES-LacZ-pA cassette flanked by FRT sites (Supplementary Fig. 2k, l).

Further breeding with a Pax7creER mouse36 to generate Pax7creER/+; _SAM_fl/fl mouse (termed _SAM_ iKO) led to permanent deletion of exons 3–4 of _SAM_ in the adult Pax7+ SCs following five

consecutive doses of tamoxifen (TM) injection in 2-month-old mouse (Fig. 2m); the successful elimination of exons 3–4 of _SAM_ was confirmed (Fig. 2n); interestingly, a truncated transcript

was generated from exons 1–2 (Fig. 2n). Consistent with what was observed in the KO mouse (Fig. 2h, i), the iKO mouse also displayed impaired regenerative ability after BaCl2 induced muscle

injury as assessed by a 21.95% decreased number of eMyHC+ 4 days after injury (Fig. 2o, p). Taken together, findings from both KO and iKO mice solidify our thinking that _SAM_ is necessary

for the timely repair of damaged skeletal muscle tissue after acute injury. _SAM_ DELETION AGGRAVATES DYSTROPHIC PHENOTYPE IN MDX MOUSE Besides acute injury by BaCl2 injection, innate

genetic defects can also provoke chronic injury-induced muscle regeneration. For example, in the widely used mouse model for Duchenne muscular dystrophy (DMD), mdx mouse displays extensive

muscle degeneration and regeneration as early as ~3 weeks of age; repetitive degeneration/regeneration cycles lead to the eventual loss of SC regenerative capacity and fatty fibrosis in old

mdx mouse37,38. To examine whether _SAM_ loss may affect chronic injury-induced regeneration in DMD, we generated _SAM_; dystrophin double KO (dKO) mouse by crossing the _SAM_ KO first mouse

 with mdx mouse (Fig. 3a). As expected, _SAM_ expression was completely depleted in freshly sorted SCs of dKO vs. control (Ctrl) mdx mice (Fig. 3b). The dKO mouse displayed no overt

difference from the Ctrl mouse (Fig. 3c) with comparable body weight during the course of 27 weeks (Supplementary Fig. 3a); TA and gastrocnemius (GAS) muscles also showed comparable weight

at 8 weeks (Supplementary Fig. 3b). However, when examined closely, smaller myofibers were more frequently observed in the TA muscles of 8 weeks old dKO mouse as measured by the 

cross-sectional area (CSA) of individual fiber (Fig. 3d). Moreover, histological examination revealed increased size of unrepaired areas (Fig. 3e), an increased number of eMyHC+ myofibers

(Fig. 3f) and increased infiltration of CD68+ macrophages (Fig. 3g) in dKO mice, suggesting loss of _SAM_ delays the muscle regeneration in limb muscles. Compared to limb muscles, mdx

diaphragm (Dia) muscle is known to exhibit a more severe dystrophic phenotype manifested by fibrosis and fatty infiltration that worsens as mice age33,39. Expectedly, the dKO mice at 6–8

months displayed the exacerbation of fibrosis as evidenced by increased Collagen I or Trichrome staining (Fig. 3h–j). Taken together, the above results suggest that loss of _SAM_ aggravates

dystrophic phenotype of mdx mice. LOSS OF _SAM_ LEADS TO SC AUTONOMOUS DEFECTS IN PROLIFERATION To further elucidate the impact of _SAM_ loss on SC activities, we tested whether _SAM_ loss

impaires MB proliferation in the KO mouse (Fig. 4). First, in vivo EdU labeling after BaCl2 injury indeed revealed a reduced percentage (12.33%) of proliferating MBs in KO vs. WT littermates

(Fig. 4a and Supplementary Fig. 4a); similar reduction (14.67%) was also observed when the assay was performed in iKO vs. Ctrl littermates (Fig. 4b). To further elucidate whether this

proliferative defect is cell-autonomous, FISCs from KO or WT mice were cultured for 2 days and a lower percentage of EdU+ cells in KO (57.01%) vs. WT (66.91%) cells was detected (Fig. 4c).

Furthermore, a significant reduction of the percentage of Pax7+MyoD+ cells was observed in KO (85.61%) vs. WT (91.78%) cells, suggesting a decline in the proliferative capacity of MBs (Fig. 

4d). Consistently, when performed on SCs isolated from iKO mouse, the same conclusion was reached; a reduced percentage of EdU+ (25.61%) or Pax7+MyoD+ cells (5.59%) was found in iKO vs. Ctrl

cells (Fig. 4e, f). Moreover, we also isolated single myofibers and performed the above assays in SCs associated with the cultured myofibers. Again, the percentage of EdU+ or Pax7+MyoD+

cells was significantly reduced in KO (10.88% and 5.35%, respectively) vs. WT cells (Fig. 4g and Supplementary Fig. 4b). In addition, MTS assay also revealed that SCs from KO muscle

displayed a declining proliferating rate compared with WT control (Supplementary Fig. 4c). Of note, the impaired proliferation in KO SCs was rescued by re-expressing a _SAM_ plasmid

(Supplementary Fig. 4d, e and Fig. 4h), pinpointing loss of _SAM_ as the cause of the deficient proliferation. The above findings supported _SAM_ loss inhibits proliferation in MBs. To

further investigate if it also has any impact on other aspects of SC activities. We first found that within 30 h after isolation, the percentages of EdU+ and Pax7+ MyoD+ cells were reduced

36.62% and 17.91%, respectively in KO vs. WT (Fig. 4i, j), indicating a possible defect at the very early activation stage. Further assessing the differentiation ability, we found the

percentage of MyoG+MyoD+ cells was increased (25.72%) in KO vs. WT SCs cultured for 3 days or myofiber-associated SCs cultured for the same period (19.87%) (Fig. 4k and Supplementary Fig. 

4f). This was further substantiated by measuring the fusion index by MF20 staining after 2 days in DM; KO cells showed a higher fusion ability (2.7 fold) than WT cells (Fig. 4l), indicating

_SAM_ loss leads to an increased propensity for differentiation, which was consistent with the finding from Fig. 1n. Lastly, the TUNEL assay did not detect differences in KO vs. WT

(Supplementary Fig. 4g) cells cultured for 2 days, suggesting _SAM_ loss may not have caused SC apoptosis. Altogether, the above results demonstrate that _SAM_ deletion causes a delay in SC

activation and proliferation but increases the propensity for precocious differentiation. Lastly, the above phenotypical changes in cells were also substantiated when RNA-seq was performed

to assess transcriptomic changes caused by _SAM_ loss. The knock-down of _SAM_ led to 250 genes down-regulated and 167 genes up-regulated in MBs (Supplementary Fig. 4h). Gene ontology (GO)

cluster analysis revealed that the down-regulated genes were enriched for GO terms including cell cycle, M phase, microtubule-based process, chromatin assembly, etc. (Supplementary Fig. 4i),

in line with the above uncovered function of _SAM_ in promoting cell proliferation. The up-regulated genes were, on the other hand, enriched for skeletal system development, muscle cell

differentiation, etc. (Supplementary Fig. 4j), which was consistent with the precocious differentiation phenotype upon _SAM_ loss. _SAM_ INTERACTS WITH SUGT1 IN MBS To dissect the molecular

mechanism underlying _SAM_ function in MBs, we sought to identify the interacting protein partners of _SAM_ considering the well-known protein-binding ability of lncRNAs that endows

themselves with many regulatory capacities40. To this end, we conducted RNA-pull down assay followed by mass spectrometry (MS) in C2C12 MBs using in vitro transcribed biotin-labeled _SAM_ or

_GFP_ transcripts13 (Fig. 5a, b). A list of proteins was identified as potential interacting partners of _SAM_, among which Sugt1 caught our attention because of its known function in

kinetochore assembly and cell mitosis10,41. Next, we confirmed the _SAM_/Sugt1 association by Western blotting following RNA pull-down. Indeed, an evident amount of Sugt1 was captured by

_SAM_, but not _GFP_ transcripts (Fig. 5c). No interaction was detected between _SAM_ and a few other known RNA-binding proteins, Hnrnpl42, Dnmt3a, and Dnmt3b15, suggesting the specificity

of the _SAM_/Sugt1 association. To further confirm their interaction, native RNA immunoprecipitation (RIP) assay was performed using an antibody against Sugt1 (Fig. 5d). A higher level (3.1

fold) of _SAM_ was pulled down by the Sugt1 antibody vs. IgG control (Fig. 5d) while several control transcripts including _Gapdh_, _β-Actin_ mRNAs, and lncRNA _Dum_15 were not retrieved.

Consistently, the co-labeling of _SAM_ by RNA-FISH and Sugt1 protein by immunofluorescence (IF) revealed an evident co-localization of _SAM_ with Flag-labeled Sugt1 in MBs (Fig. 5e).

Altogether the above results substantiated that _SAM_ specifically interacts with Sugt1 protein in MBs. In addition, we generated a series of deletion fragments of _SAM_, F1–F5 according to

the predicted secondary structure by RNAfold (Supplementary Fig. 5a) and performed RNA-pulldown assay (Supplementary Fig. 5b) to map the binding domain of _SAM_ with Sugt1. Interestingly,

both F1 and F5 fragments of _SAM_ retrieved comparable amounts of Sugt1 with the full-length transcripts. Nonetheless, the truncated transcript of exons 1–2 (containing F1 and F2) did not

seem to be functional in muscle regeneration (Fig. 2n–p). To further understand how Sugt1 and _SAM_ together partake in the regulation of ASC proliferation, we found that similar to _SAM_,

_Sugt1_ expression was also up-regulated upon SC activation at 24 h but down-regulated in differentiated cells at 96 h (Fig. 5f). Functionally, when knocked down _Sugt1_ in ASCs by two

different siRNA oligos (Fig. 5g), the proliferative ability of ASCs was reduced as shown by a decreased percentage of EdU+ cells (16.53% and 13.82%) compared to controls (Fig. 5h),

phenocopying the effect of _SAM_ loss. Moreover, the overexpression of _Sugt1_ (Supplementary Fig. 5c) fully rescued the deficient proliferation of the KO ASCs (Fig. 5i). Altogether the

above results demonstrated the functional synergism of _SAM_/Sugt1 in regulating SC proliferation. The conclusion was also substantiated when the expression dynamics and loss-of-function

assays were performed using C2C12 MBs (Supplementary Fig. 5d–g). Interestingly, unlike _SAM_ loss, Sugt1 knockdown did not seem to accelerate differentiation; instead, its loss may have

delayed differentiation as assessed by the reduced number of MyoD+MyoG+ cells compared to control cells (Supplementary Fig. 5h). To further ask how _SAM_ association regulates Sugt1, we

found that _SAM_ depletion in SC did not alter the mRNA level of _Sugt1_ (Fig. 5j) or its proper localization at kinetochores in prometaphase (Supplementary Fig. 5i). Furthermore, it did not

appear to alter the basal level of Sugt1 protein (Fig. 5k, left two lanes). However, treatment with a protein biosynthesis inhibitor, CHX, caused a marked decrease (31.4%) of Sugt1 protein

in KO (Fig. 5k, lane 4 vs. 2) whereas only 18% in WT (Fig. 5k, lane 3 vs. 1), suggesting lower stability of Sugt1 in KO cells. Consistently, in a 20 h long CHX chase experiment, the

half-life of Sugt1 protein was reduced at a faster rate upon _SAM_ knockdown, confirming _SAM_ is required for maintaining the protein stability of Sugt1 (Fig. 5l). Moreover, the decreased

Sugt1 upon _SAM_ depletion was blocked in the presence of a proteasome inhibitor, MG132 (Fig. 5m, lane 6 vs. 4), suggesting _SAM_ may stabilize Sugt1 through preventing its ubiquitination.

To further test this notion, HA-tagged ubiquitin protein was over expressed in Ctrl or sh_SAM_ MBs together with Sugt1 protein; we found an increased accumulation of poly-ubiquitinated Sugt1

in sh_SAM_ cells (Fig. 5n). Consistently, the stability of Sugt1 protein was rescued after restoring _SAM_ expression in the presence of CHX without changing its RNA level (Supplementary

Fig. 5j and k). To examine if _SAM_ stabilizing Sugt1 protein specifically occurs in MB cells, we found no decrease in Sugt1 level in primary hepatocytes isolated from WT vs. KO mouse with

or without CHX treatment (Supplementary Fig. 5l). Lastly, to further strengthen that _SAM_ promotes MB proliferation through stabilizing Sugt1 protein, we found that overexpressing the WT or

a stable mutant of Sugt1 (Sugt1-4A)12 fully rescued the deficient proliferation of _SAM_ KO cells while over-expressing a highly unstable mutant of Sugt1 (Sugt1-4E)12 failed (Fig. 5o).

_SAM_/SUGT1 REGULATE KINETOCHORE ASSEMBLY IN MBS Since Sugt1 is critical for proper kinetochore assembly during cell division10,41, we next tested whether _SAM_/Sugt1 together regulate SC

proliferation through modulating kinetochore assembly. By staining chromosomes with DAPI, centromeres with ACA and spindles with α-Tubulin, in control cells, a robust spindle structure was

preserved in metaphase cells, and bundles of microtubules were observed to terminate in kinetochores (Fig. 6a). In contrast, cells with Sugt1 knockdown exhibited disorganized spindle

structures with multipolar spindles and fragmented spindle poles frequently observed (Fig. 6a). The above phenomena were also observed in C2C12 MBs when Sugt1 was decreased (Supplementary

Fig. 6a). Altogether, our data demonstrate Sugt1 is important for proper chromosomal alignment and spindle organization and thus timely mitotic division of MBs. Next, to demonstrate that

_SAM_ functions synergistically with Sugt1, we found _SAM_ KO cells displayed evident defects in chromosome alignment and mitotic spindle formation (Fig. 6b) (Supplementary movies 1–4).

Again, this was also more frequently observed in C2C12 MBs with _SAM_ knockdown vs. control cells (Supplementary Fig. 6b), confirming _SAM_ is needed for proper chromosomal alignment and

mitotic division. To further determine if the above observed mitotic defects in _SAM_-depleted cells were due to kinetochore abnormalities, we examined kinetochore–microtubule (kt–mt)

attachments under cold treatment considering the loss of cold stable kt–mt attachments is commonly used as an indicator of kinetochore defects43. WT and KO SCs were treated on ice for 10 min

followed by α-Tubulin staining of microtubules (Fig. 6c); the fluorescence intensity was markedly decreased (20.76%) in KO vs. WT SCs, suggesting _SAM_ loss led to increased instability of

microtubules due to decreased kinetochores attaching. Consistently, when performed on C2C12, the same conclusion was reached; a reduced fluorescence intensity of microtubules was found in

sh_SAM_ MBs under cold treatment (Supplementary Fig. 6c). To further pinpoint the defect in kinetochore assembly upon _SAM_ loss, we examined the localization of Mis12 complex, since it is

known as a client of Hsp90-Sugt1 to be stabilized and targeted to the kinetochore11. As expected, by IF a lower level of fluorescent signals of Dsn1 subunit was observed at the kinetochores

in the mitotic KO vs. WT cells (Fig. 6d). We next examined the localization of Hec1 giving that as a so-called keystone complex Mis12 contributes to the localization of Ndc80 complex44;

interestingly, an over-accumulation of Hec1 kinetochore signals was detected in the KO vs. WT SCs (Fig. 6e). Similar phenomena were also observed in C2C12 MBs with decreased _SAM_ knockdown

(Supplementary Fig. 6d) despite the total level of Hec1 protein was largely unaltered (Supplementary Fig. 6e). Altogether, the above results confirmed the importance of _SAM_/Sugt1 in the

proper localization of kinetochore components. Lastly, to pinpoint it is the defective kinetochore assembly that mediates _SAM_ KO phenotype, we found that knockdown of Dsn1 or Hec1 in SCs

also delayed cell proliferation as assessed by a decreased percentage of EdU+ cells (Figs. 6f, g and Supplementary Fig. 6f, g). Meanwhile, since Akt is a known client of Sugt1 and its

phosphorylation at position 473 by Sugt1 can promote cancer cell proliferation45, we tested if it could also mediate _SAM_ effect but found that Akt p473 level was not decreased in KO vs. WT

cells (Supplementary Fig. 6h). Altogether, the above findings demonstrate that _SAM_ and Sugt1 together facilitate the assembly of kinetochore complex to ensure proper microtubule

attachment in mitotic MBs. _SAM_ deletion disrupts kinetochore assembly and thus delays the cell proliferation. Lastly, since it is believed that kinetochore disruption results in the 

mitotic arrest which is often followed by cell death46,47 or induces mitotic slippage accompanied by the production of aneuploid and cell senescence48,49, we examined the consequence of such

kinetochore defects in MBs and indeed detected an increased number of aneuploidy cells in KO vs. WT ASCs (Fig. 6h). However, no sign of cell apoptosis was detected earlier (Supplementary

Fig. 4g); SA-β-Gal staining also revealed no indication of cellular senescence (Supplementary Fig. 6i). DISCUSSION In this study, we identified and characterized the functional role of a

lncRNA, _SAM_, in regulating SC activity and muscle regeneration. Collectively, our findings suggest a model, in which _SAM_ regulates SC proliferation by binding with co-chaperon protein

Sugt1 to facilitate the kinetochore assembly during mitosis, thereby governing the fidelity of cell division (Fig. 7). We infer that _SAM_ stabilizes Sugt1 protein through direct

association; it thus facilitates the correct localization of Mis12 complex which is required for proper assembly of kinetochore and microtubule attachment during the mitotic progression of

MB cells. Loss of _SAM_ in SCs leads to disrupted cell division and delayed proliferation, thus impairs muscle regeneration after acute or chronic muscle injuries. Although initially

identified in C2C12 muscle cells through integrating RNA-seq and ChIP-seq analyses, we expanded our study to SCs to show that _SAM_ is highly enriched in activated SCs. Moreover, gain or

loss of function of _SAM_ in both C2C12 cells and ASCs altered cell proliferation. Extending the in vitro cell culture-based investigation, we provided extensive mouse genetic evidence to

characterize _SAM_ function in vivo utilizing three KO mouse models: whole-body KO, SC-specific inducible KO (iKO) and mdx; _SAM_ dKO mice. Results from using all three models consistently

supported a role for _SAM_ in regulating muscle regeneration after acute and chronic injuries. The KO-first strategy allowed us to delete _SAM_ without major disruption of the genomic

region, therefore, avoiding the complication of disrupting a potential enhancer in this region. Analyzing the KO mice led to the observation that _SAM_ is not essential for mouse survival

and fertility. However, the regeneration process of skeletal muscle after acute injury by BaCl2 injection was evidently impaired in both KO and iKO mice. Nonetheless, in both models, the

injured muscle eventually recovered completely from the injury, indicating that loss of _SAM_ delays but does not block the regeneration of skeletal muscle. In the third model, the dKO mice

displayed much more severe dystrophic phenotypes characterized by extensive fibrosis compared to the mdx controls; this could be caused by the amplified proliferative defect due to repeated

cycles of degeneration–regeneration that is typical of dystrophic muscles. Taken together, findings from using the three mouse models solidified the role of _SAM_ in regulating skeletal

muscle regeneration in vivo, which adds genetic evidence for the functionality of lncRNAs in vivo. Through identifying its interacting protein partners, we gained mechanistic insights into

how _SAM_ regulates SC proliferation. Sugt1 was identified as a specific interacting partner with _SAM_ in MBs; their association is supported by results of RNA pull-down, native RIP, and

co-localization assays. Furthermore, we showed that association with _SAM_ probably serves to stabilize Sugt1 as _SAM_ loss appeared to increase the ubiquitination level of Sugt1.

Consistently, a recent report12 demonstrated that an E3 ligase, RNF41 regulates the ubiquitination of Sugt1 in a phosphorylation-dependent manner and PHLPP1 dephosphorylates Sugt1 to prevent

it from associating with RNF41. In the future it may be worthy of the efforts to further investigate if _SAM_ may facilitate the homodimerization of Sugt1 or involve in the

dephosphorylation of Sugt1 in MBs. At the cellular level, we showed Sugt1 is required for kinetochore assembly as loss of Sugt1 in MBs led to typical defects associated with cell mitosis;

for example, cells presented pronounced defects in kinetochore–microtubule attachment, spindle formation and chromosome misalignments, which is in line with what was observed in Hela cells.

Thus, in both Hela and SCs, Sugt1 appears to exert a conserved function of regulating kinetochore assembly. Similarly, loss of _SAM_ largely photocopied the kinetochore abnormalities

observed in Sugt1-depleted cells, leading us to conclude that _SAM_ and Sugt1 synergistically regulate Mis12 targeting and kinetochore assembly to control MB proliferation. Expectedly, Dsn1

kinetochore signals were significantly decreased in _SAM_ KO cells, in line with what was observed in Hela cells when Sugt1 was depleted. According to Davies et al. 11 the degradation of

Dsn1 in Hela cells is dependent on Skp-Ub ligase thus suggesting this Ub pathway may be well functional in MB cells. Nevertheless, we observed increased accumulation of Hec1 protein at the

kinetochores upon _SAM_ loss, indicating Hec1 may not be subject to Skp-Ub degradation in MBs. Still it was shown that over-accumulation of Hec1 in mouse MEF cells caused aberrant spindle

phenotype9, suggesting the mislocalization of Hec1 is indeed detrimental to the assembly of kinetochore and microtubule attachment in the _SAM_ KO cells. It is also interesting to ponder on

the fate of the MBs with the abnormality in cell division. In many studies, kinetochore defective cells will show arrest or delay in metaphase via the spindle assembly checkpoint (SAC), or

perhaps slip out of mitosis with chromosome segregation errors increasing the frequency of senescence or apoptosis47,49. Indeed, an increased number of aneuploidy cells was detected upon

_SAM_ loss, which did not lead to evident cell apoptosis or senescence; Unlike SCs deficient in SAC which resisted differentiation50, _SAM_ loss did not seem to impede MB differentiation

propensity. In fact, precocious differentiation was observed in _SAM_ KO cells, which seems to suggest that the aneuploidy MBs in _SAM_ KO may have eventually undergone premature

differentiation. Coincidentally, Gogendeau et. al. 51 has described similar consequences in neural stem cells (NSCs) and intestine stem cells (ISCs), where they found that aneuploid NSCs do

not die by apoptosis, instead, they display G1 lengthening and undergo premature differentiation51. Intriguingly, Sugt1 loss did not cause premature differentiation. We suspect it is

possible that Sugt1 depletion had caused much more severe defects so that the cells eventually underwent apoptosis without being able to differentiate; this needs to be further investigated

using a Sugt1 KO mouse model. Alternatively, it is also likely that the differentiation function of _SAM_ may not be fully dependent on Sugt1. Altogether, our findings add to the growing

list of cellular mechanisms studied in the proliferation and differentiation of muscle stem cells and their progeny, thus enhancing our knowledge in the broad field of muscle stem cell and

regeneration. Additionally, to the lncRNA field, this will add in vivo genetic evidence for lncRNA involvement in muscle regeneration and provides new insights into the mechanisms of lncRNA

action to the growing list of lncRNA functions. METHODS MOUSE STUDIES _SAM_ KO heterozygote (_SAM_−/+) mice (C57BL/6 background) were generated in the Model Animal Research Center of the

Nanjing University (Nanjing, China). FLPeR mice were purchased in the Model Animal Research Center of the Nanjing University (Nanjing, China). _SAM_ KO strain (_SAM_ KO: _SAM_−/−, littermate

control: _SAM_+/+) were housed in our laboratory animal services center at the Chinese University of Hong Kong (CUHK). Pax7creER mice were purchased from the Jackson Laboratory. Mdx mouse

strains were purchased from the Jackson Laboratory. Pax7-nGFP mice31 were gifts from by Prof. Shahragim Tajbakhsh (Institut Pasteur). _SAM_ iKO strain (_SAM_-iKO: _SAM_fl/fl; Pax7creER/+,

littermate control: _SAM_+/+; Pax7creER/+) were obtained by crossing _SAM_ KO mice with FLPeR mice and Pax7creER mice. To induce Cre-mediated _SAM_ deletion, TM (T5648, Sigma) was injected

intraperitoneally at 2 mg per 20 g body weight for 5 days. _SAM_/mdx(dKO) strain (_SAM_-dKO: _SAM_−/−; mdx, littermate control: _SAM_+/+; mdx) were generated by crossing _SAM_ KO mice with

mdx mice. To induce acute muscle injury, 50 µl of 1.2% BaCl2 (dissolved in sterile demineralized water) was injected into TA muscle of ~2 months old mice. Muscles were harvested at

designated time points for further analysis. For EdU incorporation assay in vivo, one lower hindlimb muscle was subjected to 50 µl of 1.2% BaCl2 injection. Then 10 mM EdU was injected

intraperitoneally at 70 µl per 20 g body weight 2 days after injury, followed by FACS isolation of SCs 12 h later. Cells were then collected and fixed with 4% PFA. EdU-labeled cells were

visualized using click chemistry with an Alexa Fluor® 594 conjugated azide. Pictures were captured with a fluorescence microscope (Leica). For all animal-based experiments, at least three

pairs of littermates or age-matched mice were used. Primers for mice genotyping are listed in Supplementary Table 1. All animal experiments were performed in accordance with guidelines for

experimentation with laboratory animals set in the Chinese University of Hong Kong (CUHK) and approved by the Animal Experimentation Ethics Committee of CUHK (Ref no. 15/027/MIS-6-U). The

mice were maintained in animal room with 12 h light/12 h dark cycles, temperature (22–24 °C), humidity (40–60%) at animal facility in CUHK. CELL LINE CULTURE AND DRUG TREATMENT Mouse C2C12

MB cells (CRL-1772) were obtained from American Type Culture Collection (ATCC) and cultured in growth medium, GM (DMEM medium (12800-017, Gibco) with 10% fetal bovine serum, FBS (10270-106,

Gibco), 1% penicillin/streptomycin, P/S (15140-122, Gibco)), or differentiation medium, DM (DMEM medium with 2% horse serum (16050-114, Gibco), 1% P/S) in incubator at 37 °C. MG132 (M8699,

Sigma, 10 μΜ) and CHX (Sigma, 100 μg ml−1) were used for incubation for the indicated time. SATELLITE CELL ISOLATION AND CULTURE Hindlimb muscles from mice were digested with collagenase II

(LS004177, Worthington, 1000 units ml−1) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum,

heat-inactivated (HIHS, 26050088, Gibco), 1% P/S) before SCs were liberated by treating with Collagenase II (100 units ml−1) and Dispase (17105-041, Gibco, 1.1 unit ml−1) for 30 min. The

suspensions were passed through a 20 G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and incubated with the following primary

antibodies: Vcam1-biotin (105704, BioLegend), CD31-FITC (102506, BioLegend), CD45-FITC (103108, BioLegend), and Sca1-Alxa647 (108118, BioLegend). The Vcam1 signal was amplified with

streptavidin-PE-cy7 (405206, BioLegend) or Streptavidin-PE (554061, BD Biosciences). All antibodies were used at a dilution of 1:75. The BD FACSAria Fusion Cell Sorter (BD Biosciences) was

used for SC sorting following the manufacturer’s instructions. BD FACSDiva (version 8.0.1, BD Biosciences) software is used to manage the setup, acquisition, and analysis of flow cytometry

data. Coverslips and cultural wells were coated with poly-d-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for

at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml−1 β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S, or cultured in

differentiation medium (DM) (Ham’s F10 medium containing 2% horse serum and 1% P/S). SINGLE MYOFIBERS ISOLATION AND CULTURE Briefly, EDL muscles were dissected and digested with Collagenase

II (800 units ml−1) in DMEM medium at 37 °C for 75 min. Single myofibers were released by gentle trituration with Ham’s F-10 medium containing 10% HIHS and 1% P/S) and cultured in this

medium for designated time points. CELL PROLIFERATION, APOPTOSIS, AND CELL-CYCLE ANALYSES EdU incorporation assay was performed following the instruction of Click-iT® Plus EdU Alexa Fluor®

594 Imaging Kit (C10639, Thermo Fisher Scientific). Cells were incubated with 10 µM EdU for designated time before fixation. For MTS assay, cell growth rate was evaluated by using CellTiter

96® Aqueous One Solution Reagent Cell Proliferation Assay (MTS) kit (Promega, Madison, WI) according to the manufacturer’s instruction. Generally, the cells were incubated with MTS for 3 h

before absorbance measurement at 490 nm. Apoptosis was measured by TUNEL staining using the In-Situ Cell Death Detection Kit (Roche). For cell-cycle analysis, MBs were labeled with propidium

iodide (PI) or Hoechst 33342 (5 µg ml−1) for 45 min at 37 °C and sorted in the BD FACSVerse flow cytometer or BD FACSAria Fusion Cell Sorter. The results of cell cycle were analyzed using

the WinMDI 2.8 software. SA-Β-GALACTOSIDASE STAINING Cellular senescence was evaluated by β-galactosidase activity using β-galactosidase Senescence Kit (#9860, Cell Signaling Technology).

Briefly, cells were fixed for 15 min followed by washing in PBS twice. Then fixed cells were incubated with β-galactosidase staining solution at 37 °C in a dry incubator (no CO2) at least

overnight. The cells were then observed under a microscope for the development of blue color. CHROMOSOME SPREAD ASSAY Cells were cultured for 3 days and treated with 100 ng ml−1 nocodazole

for 3 h before harvesting. Trypsinized cell pellets were resuspended in pre-warmed hypotonic solution (75 mM KCl) and incubated for 20 min at 37 °C followed by collecting by centrifugation

for 5 min at 500 × _g_ and gently resuspended with freshly prepared fixative solution (methanol/glacial acetic acid 3:1). Cells were fixed for 30 min. Two or three drops of suspended cells

were released to pre-cold slides. The slides were then air-dried, and chromosomes were stained with DAPI. ISOLATION OF MOUSE PRIMARY HEPATOCYTES Liver tissue was isolated from mice and

finely minced followed by digestion with collagenase II (400 U ml−1) in water bath with shaking at 37 °C for 30 min. Digested tissue was mixed with a 10 ml serological pipette. The solution

was triturated for 10–15 times or until the suspension traveled up and down the pipette smoothly without clogging. The cell suspension was then filtered through 70 µm cell strainer and

centrifuged by 1300 rpm for 5 min. Cell pellet was washed twice in PBS and resuspended in culture medium (DMEM supplemented with 10% FBS, 100 U ml−1 penicillin and 100 IU ml−1 streptomycin).

Primary hepatocytes were seeded on dishes and incubated at 37 °C with 5% CO2 for 3 h. After cells had adhered (3–4 h) media was removed and replaced with fresh culture medium and continued

to culture for 3 days. PLASMIDS Full-length mouse _Sugt1_ was cloned into flag-tagged pcDNA3.1(+) vector (Life Technologies) between Kpn1 and Xbal1 sites. To construct _SAM_ expression

plasmid, full length of _SAM_ was amplified and cloned into pcDNA3.1(+) vector between Nhe1 and Kpn1 sites. Enhanced green fluorescent protein (GFP) was cloned into the XbaI site of

pcDNA3.1(+) for in vitro transcription. _SAM_ and _Sugt1_ shRNAs were cloned into pSIREN Retro Q vector (Clontech). HA-Ub plasmid is a kind gift from Prof. Zhenguo Wu (Hong Kong University

of Science and Technology, HKUST). SUGT-WT,4A,4E mutant plasmids are kind gifts from Prof. Subbareddy Maddika (Laboratory of Cell Death & Cell Survival, LCDCS, India)12 REAL-TIME PCR

Total RNAs from tissues and cells were extracted using Trizol reagent (Invitrogen) following the manufacturer’s instructions. cDNAs were prepared using HiScript® II Reverse Transcriptase Kit

(Vazyme). SYBR™ Green master mixes (Life Technologies) and Light Cycler® 480 Real-Time PCR System (Roche) were used for quantitative real-time PCR (qRT-PCR) detection. _18s_ and _Gapdh_

were used for normalization. Primers for qRT-PCR are listed in Supplementary Table 1. NATIVE RIP ASSAY Native RIP assay was performed under physiological conditions without cross-linking52,

Briefly, cell lysates were incubated overnight at 4 °C with antibody that were bound to Dynabeads protein G (Life Technologies) in NT2 buffer (50 mM Tris–HCl pH 7.4, 150,145 mM NaCl, 1 mM

MgCl2, and 0.05% NP40) containing 200 units RNaseOUT, 400 μM VRC, 10 μl of 100 mM DTT and 20 mM EDTA. Beads were then washed five times with NT2 buffer and treated with proteinase K for 30 

min at 55 °C. RNAs were then isolated using the standard Trizol (Invitrogen) protocol and analyzed by qRT-PCR. Following antibodies were used in RIP assay: mouse anti-Sugt1 (sc-81822) and

Normal mouse IgG (sc-2027). RNA PULL-DOWN ASSAY Biotinylated RNAs were prepared using Biotin RNA Labeling Mix (Roche) and T7/T3 RNA in vitro transcription kit (Ambion). Fifteen micrograms of

biotin-labeled RNAs were denatured at 90 °C for 2 min and then renatured with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl2) at RT for 20 min. Folded RNAs were mixed with 2 

mg total protein lysate and incubated with 50 μl of Streptavidin agarose beads for one hour at room temperature (RT). After the incubation, beads were washed five times using RIPA buffer (50

 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.0 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100). Binding proteins were retrieved by boiling at 100 °C with loading buffer and further

analyzed by running 10% SDS–PAGE gel according to the standard protocol. Proteins were detected by Coomassie Blue Staining using standard procedure and western blot. MASS SPECTROMETRY The

band uniquely present in the _SAM_ pull-done lane after Coomassie Blue staining was cut out and subject to LC–MS/MS analysis (Shanghai Applied Protein Technology, Shanghai, China).The MS

scan was performed with the following parameters: positive ion detection; scan range (_m_/_z_) = 300–1800; resolution = 70,000 at 200_m_/_z_ automatic gain control (AGC) target = 1e6;

maximum injection time = 50 ms; dynamic exclusion = 60 s. polypeptide and polypeptide fragments were collected according to the following parameters: after each full scan, 10 fragment maps

(MS2 scan) were collected, MS2 Activation Type was HCD, isolation window was 2_m_/_z_, second-level mass spectral resolution was 17,500 at 200_m_/_z_, collision Energy was 30 eV, and

underfill was 0.1%. The MS/MS spectra were searched with MASCOT engine (Matrix Science, version 2.2). The following option was used: peptide mass tolerance = 20 ppm, fragment mass tolerance 

= 0.1 Da, enzyme = trypsin, max missed cleavages = 2, fixed modification: carbamidomethyl (C), and variable modification: oxidation (M), acetyl (Protein N-term). The identified proteins were

retrieved from the uniport mouse database (ref. no. 73952; download time: 20130313). Ion score ≥ 20. The number of unique peptides (Unique PepCount) and CoverPercent (Cover%: the number of

detected amino acids/total number of amino acids in the protein) were used to identify proteins. In this study, one sample was analyzed once by LC–MS/MS. WESTERN BLOTTING Briefly, total

proteins from cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail, PIC (88266, Thermo Fisher Scientific) for 20 min on ice. The protein concentration was determined

using a Bradford protein assay kit (Bio-Rad). The following antibodies and dilutions were used for western blot analysis. Mouse anti-Sugt1 (1:500, sc-81822, Santa Cruz), mouse

anti-α-Tubulin (1:5000, B-5-1-2, Santa Cruz), mouse anti-Flag (1:1000, F1804, Sigma), mouse anti-Ub (1:5000, sc-8017, Santa Cruz), mouse anti-HA (1:1000, sc-7392, Santa Cruz), rabbit

anti-Hnrnpl (1:1000, sc-28726, Santa Cruz), mouse anti-Dnmt 3a (1:1000, ab-13888, Abcam); rabbit anti-Dnmt 3b (1:1000, ab-2851, Abcam); and rabbit anti-Hec1 antibody9 (1:5000) a very kind

gift from Dr. Robert Benezra, Memorial Sloan Kettering Cancer Center, USA). The relative band intensities were quantified using ImageJ 1.50i (National Institutes of Health).

IMMUNOPRECIPITATION ASSAYS Cells were lysed with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP-40). The whole-cell lysates obtained by

centrifugation (with equal concentration of protein in different samples) were incubated with 1 µg of Sugt1 antibody for overnight at 4 °C with rotation followed by binding to Dynabeads™

Protein G (Invitrogen) for 6 h at 4 °C. The immunocomplexes were then washed with washing buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, and 1% Triton X-100) four

times and applied to SDS–PAGE. IN VIVO UBIQUITINATION ASSAY C2C12 cells were transfected with HA-ubiquitin and flag-Sugt1 plasmids. 38 h after transfection, cells were treated with MG132 (10

 µM) for 10 h. The whole-cell extracts prepared by lysis buffer were subjected to immunoprecipitation of Sugt1 protein. The levels of ubiquitinated protein were then detected by

immunoblotting with HA antibody. IF STAINING AND IMAGE ACQUISITION For IF staining, cells were fixed in 4% PFA for 15 min and permeabilized with 0.5% NP-40 for 10 min. Then cells were

blocked in 5% BSA for 1 h followed by incubating with primary antibodies overnight at 4 °C and secondary antibodies for one hour at RT. For kinetochore protein staining, cells need be

pre-permeabilized in 1% Triton X-100 in PHEM buffer (60 mM Pipes, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) for 5 min before cells were fixed with 3.7% formaldehyde (Sigma) for 20 

min. After fixation, cells were proceeded as described above. For cold-stable microtubule analysis, cells were incubated on ice for indicated times followed by fixation with PHEM buffer

containing 3.7% formaldehyde and 0.2% Triton X-100 for 10 min on ice and cells then were stained as above. Antibodies and dilutions were used as following: rabbit anti-MyoD (1:100, Santa

Cruz Biotechnology, Inc); rabbit anti-MyoG (1:200, Santa Cruz Biotechnology, Inc); mouse anti-Pax7 (1:100, Developmental Studies Hybridoma Bank); mouse anti-MF20 (1:50, Developmental Studies

Hybridoma Bank); Donkey anti-Mouse IgG Alexa Fluor 488 or 594 (1:200, Invitrogen), Donkey anti-Rabbit IgG Alexa Fluor 594 (1:200, Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (1:200,

Invitrogen); mouse anti-α-Tubulin (1:400, Santa Cruz), rabbit anti-Hec1 (1:200; a very kind gift from Robert Benezra, Memorial Sloan Kettering Cancer Center, USA), rabbit anti-Dsn1 (1:100;

Biorbyt), and ACA (1:50, Antibodies Incorporated). All images were captured by a fluorescence microscope (Leica, DM 6000B) with Leica LAS AF software (LAS AF2.6.3) and laser scanning

confocal microscope (Carl ZEISS LSM 880) with ZEN 2.3 (blue edition) software. For measurements of fluorescence intensities, 10 optical slices were acquired at 0.3 μm intervals. Measurements

of tubulin, Hec1, Dsn1, and Sugt1 intensities were conducted with maximum intensity projections of images by in house program written in MATLAB (R2014b) language. Exposure settings were

held constant within each group of experiments. IMMUNOHISTOCHEMISTRY53 In brief, slides were fixed with 4% PFA for 15 min at RT and permeabilized in ice cold menthol for 6 min at −20 °C.

Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After 4% BBBSA (4% IgG-free BSA in PBS; Jackson, 001-000-162) blocking, the

sections were further blocked with unconjugated AffiniPure Fab Fragment (1:100 in PBS; Jackson, 115-007-003) for 30 min. The biotin-conjugated anti-mouse IgG (1:500 in 4% BBBSA, Jackson,

115-065-205) and Cy3-Streptavidin (1:1250 in 4% BBBSA, Jackson, 016-160-084) were used as secondary antibodies. Primary antibodies and dilutions were used as following: mouse anti-PAX7

(1:50, DSHB), mouse anti-MyoD (1:100, Dako, M3512), mouse anti-eMyHC (1:300, Leica, NCL-MHC-d), rabbit anti-Collagen1 (1:200; Novus, NBP1-30054), and rabbit anti-laminin (1:800,

Sigma-Aldrich, L9393). Masson’s trichrome staining was performed according to the manufacturer’s instructions (ScyTek Laboratories, Logan, UT). All fluorescent images were captured with a

fluorescence microscope (Leica, DM 6000B). Measurements of Collagen 1 and collagen positive area were conducted by in house ImageCount software written in MATLAB (R2014b) language. RNA

FLUORESCENCE IN SITU HYBRIDIZATION54 The Stellaris™-type oligonucleotides targeting _SAM_ were modified with Biotin. Probe sequences are shown in Supplementary Table 2. Briefly, For _SAM_

FISH, cells were fixed with 3.7% formaldehyde for 10 min at RT and permeabilized in 70% ethanol overnight at 4 °C and hybridized with probes in buffer (2× SSC, pH = 7.0, 10% formamide, 2 mM

VRC, 0.2 mg ml−1 BSA, 1 mg ml−1 yeast tRNA, and 100 mg ml−1 dextran sulfate) for overnight at 37 °C. After washing, cells were blocked with 4% BSA and then incubated with Cy3-streptavidin

antibody (Jackson, ref: 016-160). Prolong Gold antifade reagent was applied to mount the slides for DAPI. Images were taken with a ×63 NA 1.4 oil objective on the laser scanning confocal

microscope (Carl Zeiss LSM 880). For FISH and flag-Sugt1 IF co-staining, prior to the hybridization, cells fixed in 3.7% formaldehyde and stored in 70% ethanol were permeabilized with 0.5%

Triton x-100 for 10 min at RT. After washing cells were proceeded with the FISH protocol as described above. The following antibodies and dilutions were used. Mouse anti-flag (1:200, Sigma).

Goat anti–mouse IgG Alexa Fluor 488 (1:200, Invitrogen). RNA-SEQ AND DATA ANALYSIS For library construction, we used a protocol as described before13,14. The purified library products were

evaluated using a Bioanalyzer (Agilent) and SYBR qPCR and sequenced on an Illumina Hi-seq 2000 sequencer (pair-end with 50 bp). Sequenced fragments were mapped to reference mouse genome

(mm9) using TopHat254. Cufflinks55 was then used to estimate the relative abundance of transcripts in RNA-Seq experiments. Abundances were reported in fragments per kilobase per million

(FPKM), which is conceptually analogous to the reads per kilobase per million (RPKM) used for single-end RNA-seq. Differentially expressed genes were identified if the fold change ≥ 1.5 by

comparing si_SAM_ and si_NC_ samples. STATISTICS AND REPRODUCIBILITY Data were analyzed using GraphPad Prism (version 8; GraphPad Software, San Diego, CA). Data were represented as the

average of at least three biologically independent samples ± SD or ±SEM unless indicated. The statistical significance was assessed by the Student’s two-tailed paired and unpaired _t_-test.

ns, not significant. Representative images of at least three independent experiments were shown in Fig. 5a, c, d, e, k, m, n. and Supplementary Figs. 2b, e, f, l; 4g; 5d, e, 6e and h.

Representative images of two independent experiments were shown in Supplementary Fig. 5b, k, and l. REPORTING SUMMARY Further information on research design is available in the Nature

Research Reporting Summary linked to this article. DATA AVAILABILITY The data supporting the findings of this study are available from the corresponding author on reasonable request. RNA-seq

data have been deposited in the Gene Expression Omnibus under the accession code GSE126423. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via

the PRIDE56 partner repository with the dataset identifier PXD018147. The source data underlying Figs. 1c–e, 1g–n, 2b, d, f, h–k, n, p, 3b, d, f, g, i, j, 4a–l, 5a, c, d, f–o, 6a–h and

Supplementary Figs. 1b, d, f–t, 2b–d, g, h, j, l, 3a, b, 4b–f, 5b–l, 6a–i are provided in the Source Data file. CODE AVAILABILITY MATLAB language codes used in this study have been deposited

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47, D442–D450 (2019). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Prof. Robert Benezra for his generous sharing the antibody of mouse Hec1; Prof.

Subbareddy Maddika for sharing SUGT1-WT, SUGT1-4A, SUGT1-4E plasmids; Prof. Mara Brancaccio for sharing the Flag-Sugt1 plasmid; Prof. Ken Kaplan for his kind suggestions on

kinetochore-related biology; Dr. Han Zhu and Prof. Tom. H. Cheung for their suggestions on fluorescence-activated cell sorting (FACS). This work was supported by General Research Funds (GRF)

from the Research Grants Council (RGC) of the Hong Kong Special Administrative Region (14115319, 14133016, 14106117, and 14100018 to H.W.; 14116918 and 14120619 to H.S.); the National

Natural Science Foundation of China (NSFC) to H.W. (Project code: 31871304), NSFC/RGC Joint Research Scheme to H.S. (Project code: N_CUHK 413/18); Focused Innovations Scheme: Scheme B to

H.S. [Project Code: 1907307]. AUTHOR INFORMATION Author notes * Leina Lu Present address: Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University,

Cleveland, 44106, OH, USA AUTHORS AND AFFILIATIONS * Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong, China Yuying Li, Jie Yuan, Leina Lu & Hao Sun *

Department of Orthaepedics and Traumatology, The Chinese University of Hong Kong, Hong Kong, China Fengyuan Chen, Suyang Zhang, Yu Zhao, Xiaona Chen & Huating Wang * Li Ka Shing

Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China Leina Lu, Hao Sun & Huating Wang * Department of Toxicology, School of Public Health, Southern Medical

University, Guangzhou, China Liang Zhou * Department of Obstetrics and Gynaecology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China Ching Yan

Chu Authors * Yuying Li View author publications You can also search for this author inPubMed Google Scholar * Jie Yuan View author publications You can also search for this author inPubMed

 Google Scholar * Fengyuan Chen View author publications You can also search for this author inPubMed Google Scholar * Suyang Zhang View author publications You can also search for this

author inPubMed Google Scholar * Yu Zhao View author publications You can also search for this author inPubMed Google Scholar * Xiaona Chen View author publications You can also search for

this author inPubMed Google Scholar * Leina Lu View author publications You can also search for this author inPubMed Google Scholar * Liang Zhou View author publications You can also search

for this author inPubMed Google Scholar * Ching Yan Chu View author publications You can also search for this author inPubMed Google Scholar * Hao Sun View author publications You can also

search for this author inPubMed Google Scholar * Huating Wang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.L. designed and performed

most experiments, analyzed data, interpreted results, and drafted the manuscript. J.Y. performed image processing and analyzed RNA-seq data. S.Z. and F.C. performed individual mice

experiments. Y.Z. and X.C. provided support and suggestions for FISH and RNA pulldown. L.L. and L.Z. provided individual cell experiments. C.Y.C. provided technique support during FACS

isolation. H.S. and H.W. conceived the project, designed experiments, and wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Hao Sun or Huating Wang. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks Shihuan Kuang and the other, anonymous, reviewer(s) for

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Li, Y., Yuan, J., Chen, F. _et al._ Long noncoding RNA _SAM_ promotes myoblast

proliferation through stabilizing Sugt1 and facilitating kinetochore assembly. _Nat Commun_ 11, 2725 (2020). https://doi.org/10.1038/s41467-020-16553-6 Download citation * Received: 18 March

2019 * Accepted: 30 April 2020 * Published: 01 June 2020 * DOI: https://doi.org/10.1038/s41467-020-16553-6 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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