Post-transcriptional processing of some long non-coding RNAs (lncRNAs) reveals that they are a source of miRNAs. We show that the 268-nt non-coding RNA component of mitochondrial RNA processing endoribonuclease, (RNase MRP), is the source of at least two short (∼20 nt) RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs. Point mutations in RNase MRP cause human cartilage-hair hypoplasia (CHH), and several disease-causing mutations map to RMRP-S1 and -S2. SHAPE chemical probing identified two alternative secondary structures altered by disease mutations. RMRP-S1 and -S2 are significantly reduced in two fibroblast cell lines and a B-cell line derived from CHH patients. Tests of gene regulatory activity of RMRP-S1 and -S2 identified over 900 genes that were significantly regulated, of which over 75% were down-regulated, and 90% contained target sites with seed complements of RMRP-S1 and -S2 predominantly in their 3' UTRs. Pathway analysis identified regulated genes that function in skeletal development, hair development and hematopoietic cell differentiation including PTCH2 and SOX4 among others, linked to major CHH phenotypes. Also, genes associated with alternative RNA splicing, cell proliferation and differentiation were highly targeted. Therefore, alterations RMRP-S1 and -S2, caused by point mutations in RMRP, are strongly implicated in the molecular mechanism of CHH.

MicroRNAs (miRNAs) regulate cell fate selection and cellular differentiation. miRNAs of the miR23b polycistron (miR-23b, miR-27b, and miR-24) target components of the TGF-β signaling pathway and affect murine bile ductular and hepatocyte cell fate selection in vitro. Here we show that miR-23b polycistron miRNAs directly target murine Smad4, which is required for TGF-β signaling. Injection of antagomirs against these miRNAs directly into E16.5 murine fetuses caused increased cytokeratin expression in sinusoids and primitive ductular elements throughout the parenchyma of newborn mice. Similar antagomir injection in newborn mice increased bile ductular differentiation in the liver periphery and reduced hepatocyte proliferation. Antagomir injection in newborn Alb/TGF-β1 transgenic mice that develop fibrosis inhibited the development of fibrosis, and injection of older mice caused the resolution of existing fibrosis. Furthermore, murine stellate cell activation, including ColA1 and ACTA2 expression, is regulated by miR-23b cluster miRNAs. In summary, knockdown of miR-23b cluster miRNAs in fetal and newborn liver promotes bile duct differentiation and can block or revert TGF-β-induced liver fibrosis that is dependent on stellate cell activation. These data may find practical application in the highly needed development of therapies for the treatment of fibrosis.

SummaryThe organization of the genome in three-dimensional space has been shown to play an important role in gene expression. Specifically, facets of genomic interaction such as topologically associated domains (TADs) have been shown to regulate transcription by bringing regulatory elements into close proximity1. mRNA production is an intricate process with multiple control points including regulation of Pol II elongation and the removal of non-coding sequences via pre-mRNA splicing2. The connection between genomic compartments and the kinetics of RNA biogenesis and processing has been largely unexplored. Here, we measure Pol II elongation and splicing kinetics genome-wide using a novel technique that couples nascent RNA-seq with a mathematical model of transcription and co-transcriptional RNA processing. We uncovered multiple layers of spatial organization of these rates: the rate of splicing is coordinated across introns within individual genes, and both elongation and splicing rates are coordinated within TADs, as are alternative splicing outcomes. Overall, our work establishes that the kinetics of transcription and splicing are coordinated by the spatial organization of the genome and suggests that TADs are a major platform for coordination of alternative splicing.

Although expansion of snRNA genes in the human genome and sequence variation in expressed transcripts were both identified long ago, no study has comprehensively analyzed which genes are transcriptionally active. Here, we use comprehensive bioinformatic analysis to differentiate between similar or identical genomic loci to determine that 49 snRNA genes are actively transcribed. This greatly expands on previous observation of sequence variation within snRNA transcripts. Further analysis of U2 snRNA variants reveals sequence variation maintains conserved secondary structures, yet sensitizes these U2 snRNAs to modulation of assembly factors. Homeostasis of total U2 snRNA level is maintained by altering the ratio of canonical and an abundant U2 snRNA variant. Both canonical and variant snRNA promoters respond to MYC and appear differentially sensitive to increased MYC levels. Thus, we identify transcribed snRNA variants and the sequence variation within, and propose mechanisms of transcriptional and posttranscriptional regulation of snRNA levels and pre-mRNA splicing. (Summary: 150/150 words) KEYWORDS: U2 snRNA, snRNA pseudogene, spliceosome, Sm binding site HIGHLIGHTS • ChIP-seq of active promoters identifies uncharacterized snRNA genes • Transcribed repetitive snRNA genes are distinguished from falsely-mapped snRNA loci • U2 snRNA variants are sensitive to modulations in snRNP assembly • Widely expressed U2 snRNA variants provide homeostasis for total U2 snRNP levels

Protein arginine methyltransferases (PRMTs) methylate histones, splicing factors, and many other nuclear proteins. Type I enzymes (PRMT1-4,6,8) catalyze mono- (Rme1/MMA) and asymmetric (Rme2a/ADMA) dimethylation; Type II enzymes (PRMT5,9) catalyze mono- and symmetric (Rme2s/SDMA) dimethylation. Misregulation of PRMTs in multiple types of cancers is associated with aberrant gene expression and RNA splicing. To understand the specific mechanisms of PRMT activity in splicing regulation, we treated cells with the PRMT5 inhibitor GSK591 and the Type I inhibitor MS023 and probed their transcriptomic consequences. We discovered that Type I PRMTs and PRMT5 inversely regulate core spliceosomal Sm protein Rme2s and intron retention. Loss of Sm Rme2s is associated with the accumulation of polyadenylated RNA containing retained introns and snRNPs on chromatin. Conversely, increased Sm Rme2s correlates with decreased intron retention and chromatin-association of intron-containing polyadenylated RNA. Using the newly developed SKaTER-seq model, comprehensive and quantitative analysis of co-transcriptional splicing revealed that either Type I PRMT or PRMT5 inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternatively spliced mRNA. Quantitation of retained intron decay following inhibition of nascent transcription revealed that Type I PRMTs and PRMT5 reciprocally regulate post-transcriptional splicing efficiency.