Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.     

POS-1 and GLD-1 repress glp-1 translation through a conserved binding-site cluster

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.     

AGO-RBP crosstalk on target mRNAs: Implications in miRNA-guided gene silencing and cancer

MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) are important regulators of mRNA translation and stability in eukaryotes. While miRNAs can only bind their target mRNAs in association with Argonaute proteins (AGOs), RBPs directly bind their targets either as single entities or in complex with other RBPs to control mRNA metabolism. miRNA binding in 3′ untranslated regions (3′ UTRs) of mRNAs facilitates an intricate network of interactions between miRNA-AGO and RBPs, thus determining the fate of overlapping targets. Here, we review the current knowledge on the interplay between miRNA-AGO and multiple RBPs in different cellular contexts, the rules underlying their synergism and antagonism on target mRNAs, as well as highlight the implications of these regulatory modules in cancer initiation and progression.     

Genome-wide analysis of GLD-1-mediated mRNA regulation suggests a role in mRNA storage

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are "stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1-dependent repression prevents precocious translation of OET-promoting mRNAs. Secondly, GLD-1- and CGH-1-dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised.     

Global signatures of protein binding on structured RNAs in Saccharomyces cerevisiae

Protein binding is essential to the transport,decay and regulation of almost all RNA molecules.However,the structural preference of protein binding on RNAs and their cellular functions and dynamics upon changing environmental conditions are poorly understood.Here,we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins(RBPs)and structured RNAs in yeast at single-nucleotide resolution.We found that on average,in terms of percent total lengths,~15%of mRNA untranslated regions(UTRs),~37%of canonical non-coding RNAs(ncRNAs)and~11%of long ncRNAs(lncRNAs)are bound by proteins.The RBP binding sites,in general,tend to occur at single-stranded loops,with evolutionarily conserved signatures,and often facilitate a specific RNA structure conformation in vivo.We found that four nucleotide modifications of tRNA are significantly associated with RBP binding.We also identified various structural motifs bound by RBPs in the UTRs of mRNAs,associated with localization,degradation and stress responses.Moreover,we identified>200 novel lncRNAs bound by RBPs,and about half of them contain conserved secondary structures.We present the first ensemble pattern of RBP binding sites in the structured non-coding regions of a eukaryotic genome,emphasizing their structural context and cellular functions.     

Selection on Synonymous Sites for Increased Accessibility around miRNA Binding Sites in Plants

Synonymous codons are widely selected for various biological mechanisms in both prokaryotes and eukaryotes. Recent evidence suggests that microRNA (miRNA) function may affect synonymous codon choices near miRNA target sites. To better understand this, we perform genome-wide analysis on synonymous codon usage around miRNA target sites in four plant genomes. We observed a general trend of increased site accessibility around miRNA target sites in plants. Guanine-cytosine (GC)-poor codons are preferred in the flank region of miRNA target sites. Within-genome analyses show significant variation among miRNA targets in species. GC content of the target gene can partly explain the variation of site accessibility among miRNA targets. miRNA targets in GC-rich genes show stronger selection signals than those in GC-poor genes. Gene's codon usage bias and the conservation level of miRNA and its target also have some effects on site accessibility, but the expression level of miRNA or its target and the mechanism of miRNA activity do not contribute to site accessibility differences among miRNA targets. We suggest that synonymous codons near miRNA targets are selected for efficient miRNA binding and proper miRNA function. Our results present a new dimension of natural selection on synonymous codons near miRNA target sites in plants, which will have important implications of coding sequence evolution.     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.     

GLD-3, a bicaudal-C homolog that inhibits FBF to control germline sex determination in C. elegans

The FBF RNA binding proteins control multiple aspects of C. elegans germline development, including sex determination. FBF promotes the oocyte fate at the expense of spermatogenesis by binding a regulatory element in the fem-3 3'UTR and repressing this sex-determining gene. Here we report the discovery of GLD-3, a Bicaudal-C homolog and cytoplasmic protein that physically interacts with FBF. Using RNAi and a gld-3 deletion mutant, we show that GLD-3 promotes the sperm fate, a sex determination effect opposite to that of FBF. By epistasis analysis, GLD-3 acts upstream of FBF, and, in a yeast three-hybrid assay, GLD-3 interferes specifically with FBF binding to the fem-3 3'UTR. We propose that GLD-3 binds FBF and thereby inhibits its repression of target mRNAs.     

Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.