Proteins are built by using the information contained in molecules of messenger RNA (mRNA). Cells have several ways of controlling the amounts of different proteins they make. For example, a so-called ‘microRNA’ molecule can bind to an mRNA molecule to cause it to be more rapidly degraded and less efficiently used, thereby reducing the amount of protein built from that mRNA. Indeed, microRNAs are thought to help control the amount of protein made from most human genes, and biologists are working to predict the amount of control imparted by each microRNA on each of its mRNA targets. All RNA molecules are made up of a sequence of bases, each commonly known by a single letter—‘A’, ‘U’, ‘C’ or ‘G’. These bases can each pair up with one specific other base—‘A’ pairs with ‘U’, and ‘C’ pairs with ‘G’. To direct the repression of an mRNA molecule, a region of the microRNA known as a ‘seed’ binds to a complementary sequence in the target mRNA. ‘Canonical sites’ are regions in the mRNA that contain the exact sequence of partner bases for the bases in the microRNA seed. Some canonical sites are more effective at mRNA control than others. ‘Non-canonical sites’ also exist in which the pairing between the microRNA seed and mRNA does not completely match. Previous work has suggested that many non-canonical sites can also control mRNA degradation and usage. Agarwal et al. first used large experimental datasets from many sources to investigate microRNA activity in more detail. As expected, when mRNAs had canonical sites that matched the microRNA, mRNA levels and usage tended to drop. However, no effect was observed when the mRNAs only had recently identified non-canonical sites. This suggests that microRNAs primarily bind to canonical sites to control protein production. Based on these results, Agarwal et al. further developed a statistical model that predicts the effects of microRNAs binding to canonical sites. The updated model considers 14 different features of the microRNA, microRNA site, or mRNA—including the mRNA sequence around the site—to predict which sites within mRNAs are most effectively targeted by microRNAs. Tests showed that Agarwal et al.'s model was as good as experimental approaches at identifying the effective target sites, and was better than existing computational models. The model has been used to power the latest version of a freely available resource called TargetScan, and so could prove a valuable resource for researchers investigating the many important roles of microRNAs in controlling protein production.

/pdf/predicting-effective-microrna-target-sites-in-mammalian-4ivmgdccgc.pdf

Predicting effective microRNA target sites in mammalian mRNAs

MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.

Regulation of microRNA biogenesis

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are ~21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein-protein and protein-RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

http://m.docente.unife.it/franco.pagani/lezione-1101a-mirna/articoli-smallrna/2010%20FIlipowic%20The%20widespread%20regulation%20of%20miRNA%20%20copy.pdf

The widespread regulation of microRNA biogenesis, function and decay.

miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

/pdf/mirbase-integrating-microrna-annotation-and-deep-sequencing-3rcas34b0z.pdf

miRBase: integrating microRNA annotation and deep-sequencing data

microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically downregulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers.

/pdf/mirdeep2-accurately-identifies-known-and-hundreds-of-novel-22e5p7gok3.pdf

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

/pdf/mammalian-micrornas-experimental-evaluation-of-novel-and-3o8h3xwfet.pdf

Lori W. Schoenfeld

Papers

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes