Summary Transcription of the mammalian genome is pervasive but productive transcription outside protein-coding genes is limited by unknown mechanisms1. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense coding direction2–4. Here we show that asymmetric sequence determinants flanking gene transcription start sites (TSS) control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs (uaRNAs) are cleaved and polyadenylated at poly (A) sites (PAS) shortly after their initiation. De novo motif analysis reveals PAS signals and U1 snRNP (U1) recognition sites as the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 and PAS sites are progressively gained and lost, respectively, at the 5′ end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a significant increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggests that a U1-PAS axis characterized by low U1 recognition and high density of PAS in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.
Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.development | expression T he non-protein-coding portion of the mammalian genome is transcribed into a vast array of RNA species (1), some of which play important roles in cellular regulation, development, and disease (2). The long noncoding RNAs (lncRNAs) are of particular interest because they are known to contribute to gene silencing (3), X inactivation (4), imprinting (5, 6), and development (7-9), but there is limited understanding of the genomic origin, regulation, and function of lncRNA molecules in individual cell types.Embryonic stem cells (ESCs) are widely used as a model system to study transcriptional control of cell state during early development (10-13), yet there is no catalog of lncRNAs in human (h) ESCs, and it is not clear how lncRNAs are regulated in these cells. Catalogs of lncRNAs have been recently described in various murine (14, 15) and human cell types (16)(17)(18)(19), but the majority were limited to spliced lncRNA species (14-16, 18) and those distant from protein-coding genes (14-17). Because lncRNAs tend to be cell-type-specific (16, 18), these catalogs likely contain only a very small fraction of lncRNAs expressed in hESCs.We describe here catalogs of human and murine ESC lncRNAs and the genomic regions from which these RNA species arise. We find that the majority of these lncRNAs originate from divergent transcription of lncRNA/mRNA gene pairs and that many such gene pairs are coordinately regulated when ESCs differentiate.Results lncRNAs Expressed in Human ESCs. We compiled a catalog of lncRNA species expressed in hESCs as summarized in Fig. 1A. An initial pool of RNA candidates was generated by sequencing polyadenylated RNA species from hESCs and supplementing these with EST data from the full-length long Japan (FLJ) collection of sequenced human cDNAs, which contains transcripts expressed in >60 human tissues, including embryonal tissue (20). An initial pool of 170,162 ncRNA candidates (Dataset S1) was obtained after removing protein-coding transcripts based on the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq). This pool was further filtered by using multiple criteria to identify lncRNAs. The RNA species were required to have a 5′ end that originates from a genomic site where there is corroborating evidence of active transcript...
The addition of creatine to the glucose/taurine/electrolyte supplement promoted greater gains in fat/bone-free mass, isotonic lifting volume, and sprint performance during intense resistance/agility training.
Satellite cells are adult myogenic stem cells that function to repair damaged muscle. The enduring capacity for muscle regeneration requires efficient satellite cell expansion after injury, differentiation to produce myoblasts that can reconstitute damaged fibers, and self-renewal to replenish the muscle stem cell pool for subsequent rounds of injury and repair. Emerging studies indicate that misregulations of satellite cell fate and function contribute to age-associated muscle dysfunction and influence the severity of muscle diseases, including Duchenne Muscular Dystrophy (DMD). It has also become apparent that satellite cell fate during muscle regeneration, aging, and in the context of DMD is governed by an intricate network of intrinsic and extrinsic regulators. Targeted manipulation of this network may offer unique opportunities for muscle regenerative medicine.
Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5′ termini and heterogeneous nonpolyadenylated 3′ ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1-4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome.non-coding RNA | polymerase pausing R NA polymerase II (RNAPII) transcription is a highly regulated process controlling cell type and state. Recruitment of chromatin modifying factors and RNAPII to promoters by DNA binding transcription factors are key regulatory steps (1-3). However, genome-wide profiling of RNAPII indicates that this polymerase is bound and engaged in the early steps of transcriptional initiation at most active and many inactive genes in human embryonic stem cells suggesting postinitiation modes of regulation may occur more frequently than previously appreciated (4). Moreover, divergent transcription, defined by detection of populations of low abundance small RNAs (19-25 nts) generated by nonoverlapping (separated by approximately 250 bps) bidirectional transcription, was detected at the majority of transcriptional start sites (TSSs) in mouse embryonic stem cells (mESCs) (5). Polymerases engaged in divergent transcription near promoters were simultaneously described in human lung fibroblasts (6). Surprisingly, RNAPII only productively elongates in the proteincoding sense direction from these divergent promoters. Related results have been reported for several other eukaryotic systems (7-11). Altogether, these data suggest that control of RNAPII elongation and RNA stability may be major points of transcriptional regulation and that mechanisms controlling these processes may dictate whether a stable RNA molecule is synthesized.In recent years it has become clear that RNAPII pausing is a major mode of transcriptional regulation (6, 12). The Negative Elongation Factor (NELF) and DRB-Sensitivity Inducing Factor (D...
Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.
Using 42 strength and functional assessments recorded monthly, the natural history of amyotrophic lateral sclerosis (ALS) is described in 167 patients (98 men, 67 women) followed in five medical centers in the western United States. The mean age at onset was 57.4 years, and symptoms were present for 2.64 years before study entry. Although there was a highly variable rate of decline within the group of patients, there were no differences in rate of decline by age or gender. Older patients and women were weaker on entry. Forty-eight patients died during the study. The median survival was 4.0 years for the study cohort but 2.1 years for newly diagnosed cases. Decline in pulmonary function most closely correlated with death. Our results emphasize the importance of considering clinical variability in planning clinical trials. One possible strategy is to identify and stratify patients by rate of decline in pulmonary function since prospectively identifying homogeneous subgroups allows investigators to substantially reduce sample size in therapeutic trials.
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