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

AbstractTranscription 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

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

MicroRNAs in the Making: Post-transcriptional Regulation of MicroRNA Biogenesis in Caenorhabditis elegans

MicroRNAs (miRNAs) are small non-coding RNAs, ~22 nucleotides (nt) long, with major roles in gene regulation. MiRNAs bind imperfectly to complementary sequences in the 3’ untranslated region of target messenger RNAs (mRNAs) causing translational repression and destabilization. A single miRNA has the potential to regulate hundreds of different mRNA targets, highlighting the importance of miRNAs in almost all cellular pathways. Originally discovered as part of the Caenorhabditis elegans (C. elegans) developmental timing pathway, miRNAs were soon found in a multitude of other organisms, including humans. MiRBase, an online database for miRNAs, now lists >35,000 miRNAs, in >200 different species, including viruses, though many of their roles remain to be characterized. Because misregulation is often associated with disease, especially cancer, exploring miRNA biogenesis is critical for understanding the intricacies of disease development. Furthermore, the conserved temporal expression of various miRNAs highlights the importance of these regulators in pluripotency and development. Understanding how miRNAs are produced and regulated has been a major topic of study over the past 15 years. While the basic mechanisms of miRNA biogenesis and function have been uncovered, how these processes are regulated remains an outstanding problem.There are multiple instances of transcriptional and post-transcriptional regulation during miRNA biogenesis. In Chapter I, I introduce much of the latest understanding about the mechanisms of miRNA biogenesis and regulation. Details about the discovery of miRNAs, C. elegans as a model, as well as general information on biogenesis and targeting, can be found in Chapter II. I worked on several projects investigating post-transcriptional regulation of miRNA biogenesis in C. elegans. In Chapter III, I identify and characterize the primary lin-4 transcripts, and demonstrate how a conserved RNA binding protein, RBM-28, regulates mature lin-4 expression, but not primary or precursor. My investigation in Chapter IV led to notable insights on how splicing pri-let-7 leads to a secondary structure rearrangement that facilities recognition by the Microprocessor. My survey of polycistronic worm miRNAs discussed in Chapter V indicates that there are many more examples awaiting further study. Overall, this research describes novel examples of post-transcriptional regulation of miRNAs

MicroRNAs in the Making: Post-transcriptional Regulation of MicroRNA Biogenesis in Caenorhabditis elegans

MicroRNAs (miRNAs) are small non-coding RNAs, ~22 nucleotides (nt) long, with major roles in gene regulation. MiRNAs bind imperfectly to complementary sequences in the 3’ untranslated region of target messenger RNAs (mRNAs) causing translational repression and destabilization. A single miRNA has the potential to regulate hundreds of different mRNA targets, highlighting the importance of miRNAs in almost all cellular pathways. Originally discovered as part of the Caenorhabditis elegans (C. elegans) developmental timing pathway, miRNAs were soon found in a multitude of other organisms, including humans. MiRBase, an online database for miRNAs, now lists >35,000 miRNAs, in >200 different species, including viruses, though many of their roles remain to be characterized. Because misregulation is often associated with disease, especially cancer, exploring miRNA biogenesis is critical for understanding the intricacies of disease development. Furthermore, the conserved temporal expression of various miRNAs highlights the importance of these regulators in pluripotency and development. Understanding how miRNAs are produced and regulated has been a major topic of study over the past 15 years. While the basic mechanisms of miRNA biogenesis and function have been uncovered, how these processes are regulated remains an outstanding problem.There are multiple instances of transcriptional and post-transcriptional regulation during miRNA biogenesis. In Chapter I, I introduce much of the latest understanding about the mechanisms of miRNA biogenesis and regulation. Details about the discovery of miRNAs, C. elegans as a model, as well as general information on biogenesis and targeting, can be found in Chapter II. I worked on several projects investigating post-transcriptional regulation of miRNA biogenesis in C. elegans. In Chapter III, I identify and characterize the primary lin-4 transcripts, and demonstrate how a conserved RNA binding protein, RBM-28, regulates mature lin-4 expression, but not primary or precursor. My investigation in Chapter IV led to notable insights on how splicing pri-let-7 leads to a secondary structure rearrangement that facilities recognition by the Microprocessor. My survey of polycistronic worm miRNAs discussed in Chapter V indicates that there are many more examples awaiting further study. Overall, this research describes novel examples of post-transcriptional regulation of miRNAs

Lesiones de mucosa oral relacionadas con el hábito de consumo de cigarrillo

Tesis (Odontólogo). -- Universidad de Cartagena. Facultad de Odontología. Departamento de Investigación, 2016Este trabajo busca determinar la relación entre lesiones de la mucosa bucal y el hábito de consumo de cigarrillo en un grupo de adultos que asisten a consulta odontológica en la universidad de Cartagena

The RNAi hypersensitivity of <i>mir-35-41</i>(<i>gk26</i>2) is comparable to that of <i>lin-35(n745)</i>.

<p>L4 staged worms of each strain were transferred to <i>unc-22</i> RNAi and the percentage of unaffected, twitching or paralyzed adult worms was scored 28 hours later. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are mean ± standard deviation (average of at least 2 experiments). Numbers in parentheses represent the total number of worms scored.</p

The RNAi hypersensitivity of <i>mir-35-41</i> mutants is independent of <i>nrde-3</i> activity.

<p>(A) Fluorescent microscopy showing subcellular localization of GFP::NRDE-3 in seam cells of the indicated genotypes. Pictures are representative of 50 worms analyzed for each strain. (B) Histogram representing the percentages of paralyzed worms for the indicated strains fed <i>unc-22</i> dsRNA for 28 hours from the L4 to adult stage. The means and standard deviations from three independent experiments are graphed.</p

Decreased LIN-35/Rb contributes to the RNAi hypersensitivity of <i>mir-35-41(gk262)</i> worms.

<p>(A) Northern blot analyses of <i>lin-35/Rb</i> mRNA levels in WT, <i>mir-35-41(gk262)</i> and <i>lin-35(n745)</i> embryos. After normalization to actin mRNA the average and standard deviation from 3 independent experiments was calculated with wild type levels set to one. (B) LIN-35 protein is decreased in <i>mir-35-41(gk262)</i> embryos compared to wild type, as shown by western blotting. After normalization to tubulin the average and standard deviation from 4 independent experiments was calculated with wild type levels set to one (Student's t-test *p<4×10⁻⁵). (C) PAGE Northern blot analysis of RNA from wild type and <i>lin-35(n475)</i> embryos shows similar levels of pre- and mature miR-35 expression. The rRNAs are shown as loading controls. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are representative of 3 independent experiments. (D) The indicated worm strains were grown on bacteria expressing <i>unc-22</i> dsRNA. <i>Ex[lin-35; sur-5::GFP]</i> is an extrachromosomal array that expresses <i>lin-35</i> in GFP positive (+) worms. <i>Int[sur-5::GFP]</i> is an integrated array that expresses GFP in all worms and <i>Ex[myo-2::GFP]</i> is an extrachromosomal array that expresses GFP in worms that inherit the array. Phenotype was scored as percent of twitching or paralyzed worms after 28 h of exposure to RNAi from the L4 to adult stage. Error bars represent the standard error of the mean (s.e.m) for at least two independent experiments.</p

The <i>mir-35-41(gk262)</i> mutant worms show enhanced RNAi in multiple tissues and stages of development.

<p>Worm strains of the indicated genotype were grown on bacteria expressing dsRNA against the indicated genes at 20°C. RNAi phenotype results are mean ± standard deviation (average of 3 independent experiments). Numbers in parentheses represent the total number of adult worms or embryos scored.</p>*<p>L4 staged worms were transferred to RNAi and the percentage of paralyzed (%Prl) adult worms was scored 28 hours later.</p>†<p>L1 staged worms were plated on RNAi and the percentage of adult worms showing multivulva (%Muv) or roller (% Rol) phenotypes was scored.</p>††<p>L4 staged worms were transferred to RNAi, allowed to lay embryos and adult worms were removed 28 hours later. Percent embryonic lethal (%Emb) was calculated as the number of embryos that did not hatch.</p

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 <i>in vitro</i> 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 <i>via</i> homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in <i>Saccharomyces cerevisiae</i>. 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 10¹³. 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