Global Analyses of the Effect of Different Cellular Contexts on MicroRNA Targeting

MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development—with its resulting diversity of cellular contexts—impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3′ UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3′ UTR isoforms revealed that they influence ~10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3′ UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3′ UTR landscape helps predict targeting efficacy and explain differential regulation that is observed.Korea (South). Ministry of Education, Science and Technology (MEST) (National Research Foundation of Korea. NRF-2013R1A1A1010185)National Institutes of Health (U.S.) (Grant RO1 GM067031)National Institutes of Health (U.S.) (Grant K99 GM102319)National Science Foundation (U.S.). Graduate Research Fellowship Progra

The influence of microRNAs and poly(A) tail length on endogenous mRNA–protein complexes

Background: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. Results: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. Conclusions: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.National Institutes of Health (U.S.) (Grant K99GM102319)National Institutes of Health (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Grant R01GM067031)National Institutes of Health (U.S.) (Grant R35GM118135)Natural Sciences and Engineering Research Council of Canada (Discovery Grant

Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted

Buffering of transcription rate by mRNA half-life is a conserved feature of Rett syndrome models

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the transcriptional modulator MECP2. Here, the authors measured transcription rate and mRNA half-life changes in RTT patient-derived neurons to show transcription rate buffered by mRNA half-life changes

Decapping is preceded by 3' uridylation in a novel pathway of bulk mRNA turnover.

Both end structures of eukaryotic mRNAs, namely the 5' cap and 3' poly(A) tail, are necessary for transcript stability, and loss of either is sufficient to stimulate decay. mRNA turnover is classically thought to be initiated by deadenylation, as has been particularly well described in Saccharomyces cerevisiae. Here we describe two additional, parallel decay pathways in the fission yeast Schizosaccharomyces pombe. First, in fission yeast mRNA decapping is frequently independent of deadenylation. Second, Cid1-dependent uridylation of polyadenylated mRNAs, such as act1, hcn1 and urg1, seems to stimulate decapping as part of a novel mRNA turnover pathway. Accordingly, urg1 mRNA is stabilized in cid1Delta cells. Uridylation and deadenylation act redundantly to stimulate decapping, and our data suggest that uridylation-dependent decapping is mediated by the Lsm1-7 complex. As human cells contain Cid1 orthologs, uridylation may form the basis of a widespread, conserved mechanism of mRNA decay

MicroRNA Destabilization Enables Dynamic Regulation of the miR-16 Family in Response to Cell-Cycle Changes

The miR-16 family, which targets genes important for the G1-S transition, is a known modulator of the cell cycle, and members of this family are often deleted or downregulated in many types of cancers. Here, we report the reciprocal relationship—that of the cell cycle controlling the miR-16 family. Levels of this family increase rapidly as cells are arrested in G0. Conversely, as cells are released from G0 arrest, levels of the miR-16 family rapidly decrease. Such rapid changes are made possible by the unusual instabilities of several family members. The repression mediated by the miR-16 family is sensitive to these cell-cycle changes, which suggests that the rapid upregulation of the miR-16 family reinforces cell-cycle arrest in G0. Upon cell-cycle re-entry, the rapid decay of several members allows levels of the family to decrease, alleviating repression of target genes and allowing proper resumption of the cell cycle.National Institutes of Health (U.S.) (grant GM06703)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award (GM0888))Canadian Institutes of Health Researc