Splicing remodels the let-7 primary microRNA to facilitate Drosha processing in Caenorhabditis elegans.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that use partial base-pairing to recognize and regulate the expression of messenger RNAs (mRNAs). Mature miRNAs arise from longer primary transcripts (pri-miRNAs) that are processed to a shorter hairpin precursor miRNA (pre-miRNA) by the Microprocessor complex. In Caenorhabditis elegans the primary let-7 (pri-let-7) transcript undergoes trans-splicing, where pri-let-7 is cleaved at a 3' splice site and the splice-leader-1 (SL1) sequence is appended at the 5' end. Here we investigate the role of this splicing event in the biogenesis of let-7 miRNA. We hypothesized that splicing changes the secondary structure of the pri-let-7 transcript, creating a more favorable substrate for recognition by the Microprocessor. Supporting this idea, we detected conspicuous structural differences between unspliced and SL1-spliced pri-let-7 transcripts using in vitro ribonuclease (RNase) assays. Through the generation of transgenic worm strains, we found that the RNA secondary structure produced by splicing, as opposed to the act of splicing itself, optimizes processing of pri-let-7 by the Microprocessor in vivo. We also observed that the endogenous spliced, but not the unspliced, pri-let-7 transcripts bind to the Microprocessor and accumulate upon its depletion. We conclude that splicing is a key step in generating pri-let-7 transcripts with a structure that enables downstream processing events to produce appropriate levels of mature let-7.

The miR-35-41 family of microRNAs regulates RNAi sensitivity in Caenorhabditis elegans.

RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo-RNAi) or natural endogenous (endo-RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo-RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo-siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.

Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans.

Transcription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth.

Idiosyncrasies of Viral Noncoding RNAs Provide Insights into Host Cell Biology.

Like their host cells, many viruses express noncoding RNAs (ncRNAs). Despite the technical challenge of ascribing function to ncRNAs, diverse biological roles for virally expressed ncRNAs have been described, including regulation of viral replication, modulation of host gene expression, host immune evasion, cellular survival, and cellular transformation. Insights into conserved interactions between viral ncRNAs and host cell machinery frequently lead to novel findings concerning host cell biology. In this review, we discuss the functions and biogenesis of ncRNAs produced by animal viruses. Specifically, we describe noncanonical pathways of microRNA (miRNA) biogenesis and novel mechanisms used by viruses to manipulate miRNA and messenger RNA stability. We also highlight recent advances in understanding the function of viral long ncRNAs and circular RNAs.

Let's make it happen: the role of let-7 microRNA in development.

Noncoding RNAs have emerged as an integral part of posttranscriptional gene regulation. Among that class of RNAs are the microRNAs (miRNAs), which posttranscriptionally regulate target mRNAs containing complementary sequences. The broad presence of miRNAs in lower eukaryotes, plants, and mammals highlights their importance throughout evolution. MiRNAs have been shown to regulate many pathways, including development, and disruption of miRNA function can lead to disease (Ivey and Srivastava, 2010; Jiang et al., 2009). Although the first miRNA genes were discovered in the nematode, Caenorhabditis elegans, almost 20 years ago, the field of miRNA research began when they were found in multiple organisms a little over a decade ago (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lee et al., 1993; Pasquinelli et al., 2000; Wightman et al., 1993). Here, we review one of the first characterized miRNAs, let-7, and describe its role in development and the intricacies of its biogenesis and function.

A Heritable Recombination system for synthetic Darwinian evolution in yeast.

Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology.