SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo.

SUMMARY The p53-regulated long non-coding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a co-activator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted co-activator for p53-mediated p21 expression.

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...

Argonaute (Ago) proteins mediate post-transcriptional gene repression by binding guide microRNAs (miRNAs) to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner. Here, we report the small RNA profile of Ago-deficient cells and show that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, we identified a new Ago-dependent class of non-canonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II complexes produce hairpin RNAs that are processed in a Dgcr8/Drosha-independent, but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following over-expression of mRNA constructs. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation.

The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.