Post-translational Modifications And Rna-binding Proteins

RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.90729731

Pairing beyond the Seed Supports MicroRNA Targeting Specificity

To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo

Deep sequencing identifies new and regulated microRNAs in Schmidtea mediterranea

MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed in either sexual or asexual animals. Finally, we identified five miRNAs whose expression is sensitive to γ-irradiation, suggesting they are expressed in neoblasts or early neoblast progeny. Together, these results increase the known repertoire of S. mediterranea miRNAs and identify numerous regulated miRNAs that may play important roles in regeneration, homeostasis, neoblast function, and reproduction

Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development

Suppression of supernumerary seam cell nuclei in <i>let-7</i> mutants.

<p>(A) While wild-type worms have 16 seam cell nuclei, <i>let-7(n2853)</i> worms have significantly more (∼23) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hayes1" target="_blank">[82]</a>. To score for suppression of the extra seam cell phenotype, <i>let-7</i> mutants expressing nuclear GFP in seam cells (<i>let-7(n2853)</i>;<i>Int[scm::GFP]</i>) were grown at the restrictive temperature (25<b>°</b>C) on bacteria expressing dsRNA against candidate targets and the vector control. The number of seam cell nuclei was counted in a population of 20 worms evaluated against the same size population concurrently grown on the empty vector control. RNAi clones that resulted in worm populations with significantly lower seam cell numbers (p<0.05) were retested and scored using a population of at least 20 worms. (B) Suppressors of the extra seam cell nuclei phenotype in <i>let-7(n2853)</i> (p-value<0.05) are shown by bubble plot. Each bubble indicates the number of seam cell nuclei per worm for a population (n≥20) and the size of each bubble is proportional to the number of the animals in the population with a given seam cell number. * p<0.05, ** p<0.01, *** p<0.0001 in two independent trials.</p

Argonaute associates with targets in a <i>let-7</i>–dependent manner.

<p>(A) Sequences in the indicated genes were detected by semi-quantitative PCR of cDNA from ALG-1 immunoprecipitation assays from L4 staged WT and <i>let-7(n2853)</i> strains. Based on enrichment in the WT compared to <i>let-7</i> RIP from 4 independent experiments, three new targets were identified, <i>T27D12.1</i>, <i>prmt-1</i>, and <i>opt-2</i>. (B) let-7 complementary sites (LCS) are present in each of the newly identified targets. Each LCS is within an ALG-1 binding site. (C) qPCR analysis of WT and <i>let-7(n2853)</i> cDNA from L4 staged worms. Targets were normalized to 18S ribosomal RNA. Shown is the average and standard deviation from 3 independent experiments.</p

Novel suppressors of vulval rupture in <i>let-7</i> null mutants.

<p>(A) Null <i>let-7(mn112)</i> worms were maintained with an extrachromosomal rescuing transgene (let-7(+)) co-expressing a pharyngeal GFP marker (myo-2::GFP). Progeny that lack the transgene rupture from the vulva and die. (B) The <i>let-7(mn112); Ex[let-7(+);myo-2::GFP]</i> strain was grown on bacteria expressing dsRNA corresponding to candidate targets and the empty vector control. The percent rupture of non-rescued (non-GFP) animals was determined for each RNAi clone. (C) The vector control RNAi fails to suppress vulval rupturing, while knockdown of a known target (<i>daf-12</i>) or a novel candidate (<i>sox-2</i>) allows <i>let-7(mn112)</i> animals to survive to adulthood. (D) The rate of vulval rupture was plotted for each RNAi clone tested. Green points indicate clones that reduced the rupture rate to below 75% in 2/2 experiments (n>50 worms/experiment). Purple points indicate RNAi clones depicted in (C). Red points indicate clones that failed to reproducibly meet the 75% cut-off. The vector negative controls are shown in black.</p

Genes down-regulated more than 2-fold in <i>let-7(n2853)</i> compared to wild-type.

1<p>Sequence names from WormBase (<a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>).</p>2<p>FDR corrected.</p>3<p>W = mirWIP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hammell1" target="_blank">[41]</a>, P = PITA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Kertesz1" target="_blank">[40]</a>, Y = (this study), T = TargetScan <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lewis2" target="_blank">[69]</a>, R = RNA22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Miranda1" target="_blank">[39]</a>, G = MicroTarget <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Grosshans1" target="_blank">[11]</a>, M = Miranda <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Enright1" target="_blank">[36]</a>, C = PicTar <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lall1" target="_blank">[38]</a>.</p

Phenotypic suppressors of let-7 mutants.

1<p>Sequence names from WormBase (<a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>).</p>2<p>W = mirWIP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Hammell1" target="_blank">[41]</a>, P = PITA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Kertesz1" target="_blank">[40]</a>, Y = (this paper), T = TargetScan <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lewis2" target="_blank">[69]</a>, R = RNA22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Miranda1" target="_blank">[39]</a>, G = MicroTarget <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Grosshans1" target="_blank">[11]</a>, M = Miranda <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Enright1" target="_blank">[36]</a>, C = PicTar <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Lall1" target="_blank">[38]</a>.</p>3<p>R = Suppression of rupturing phenotype (% non-rupture), S = Suppression of the extra seam cell nuclei phenotype (significance level.</p>4<p>Locations of ALG-1 binding sites C =  coding region, I = intron, 3 = 3' UTR <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003353#pgen.1003353-Zisoulis1" target="_blank">[66]</a>.</p