Control of noncoding RNA production and histone levels by a 5' tRNA fragment.

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs," are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF-5' tRF-Gly-GCC, or tRF-GG-as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs-snoRNAs, scaRNAs, and snRNAs-that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3' UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5' tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.

Multi-modal regulation of C. elegans hermaphrodite spermatogenesis by the GLD-1-FOG-2 complex.

Proper germ cell sex determination in Caenorhabditis nematodes requires a network of RNA-binding proteins (RBPs) and their target mRNAs. In some species, changes in this network enabled limited XX spermatogenesis, and thus self-fertility. In C. elegans, one of these selfing species, the global sex-determining gene tra-2 is regulated in germ cells by a conserved RBP, GLD-1, via the 3' untranslated region (3'UTR) of its transcript. A C. elegans-specific GLD-1 cofactor, FOG-2, is also required for hermaphrodite sperm fate, but how it modifies GLD-1 function is unknown. Germline feminization in gld-1 and fog-2 null mutants has been interpreted as due to cell-autonomous elevation of TRA-2 translation. Consistent with the proposed role of FOG-2 in translational control, the abundance of nearly all GLD-1 target mRNAs (including tra-2) is unchanged in fog-2 mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss of gld-1 function elevates germline TRA-2 expression to detectable levels, but loss of fog-2 function does not. A simple quantitative model of tra-2 activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly, fog-2 and gld-1 activity enable the sperm fate even when GLD-1 cannot bind to the tra-2 3' UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites within tra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as "hyper-repressors" of tra-2 activity. These variants bind RNA more weakly in vitro than does wild-type GLD-1. These results indicate that gld-1 and fog-2 regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in the C. elegans hermaphrodite is far from complete.

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using library MosSCI.

BACKGROUND: In C. elegans, germline development and early embryogenesis rely on posttranscriptional regulation of maternally transcribed mRNAs. In many cases, the 3' untranslated region (UTR) is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3'UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. RESULTS: In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RNA-binding proteins that control the expression pattern of five different maternal mRNAs. CONCLUSIONS: The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. Developmental Dynamics 245:925-936, 2016. © 2016 Wiley Periodicals, Inc.

RNA recognition by the Caenorhabditis elegans oocyte maturation determinant OMA-1.

Maternally supplied mRNAs encode proteins that pattern early embryos in many species. In the nematode Caenorhabditis elegans, a suite of RNA-binding proteins regulates expression of maternal mRNAs during oogenesis, the oocyte to embryo transition, and early embryogenesis. To understand how these RNA-binding proteins contribute to development, it is necessary to determine how they select specific mRNA targets for regulation. OMA-1 and OMA-2 are redundant proteins required for oocyte maturation--an essential part of meiosis that prepares oocytes for fertilization. Both proteins have CCCH type tandem zinc finger RNA-binding domains. Here, we define the RNA binding specificity of OMA-1 and demonstrate that OMA-1/2 are required to repress the expression of a glp-1 3'-UTR reporter in developing oocytes. OMA-1 binds with high affinity to a conserved region of the glp-1 3'-UTR previously shown to interact with POS-1 and GLD-1, RNA-binding proteins required for glp-1 reporter repression in the posterior of fertilized embryos. Our results reveal that OMA-1 is a sequence-specific RNA-binding protein required to repress expression of maternal transcripts during oogenesis and suggest that interplay between OMA-1 and other factors for overlapping binding sites helps to coordinate the transition from oocyte to embryo.

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions.

Interactions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using 'protein-centered' (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest-in a 'gene-centered' manner-yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA 'bait' can be tested versus multiple RBP 'preys' in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3' UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA.

The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.

Structure and function of nematode RNA-binding proteins.

RNA-binding proteins are critical effectors of gene expression. They guide mRNA localization, translation, and stability, and potentially play a role in regulating mRNA synthesis. The structural basis for RNA recognition by RNA-binding proteins is the key to understand how they target specific transcripts for regulation. Compared to other metazoans, nematode genomes contain a significant expansion in several RNA-binding protein families, including Pumilio-FBF (PUF), TTP-like zinc finger (TZF), and Argonaute-like (AGO) proteins. Genetic data suggest that individual members of each family have distinct functions, presumably due to sequence variations that alter RNA-binding specificity or protein interaction partners. In this review, we highlight example structures and identify the variable regions that likely contribute to functional divergence in nematodes.