The Enigmatic Conservation of a Rap1 Binding Site in the Saccharomyces cerevisiae HMR-E Silencer

Silencing at the HMR and HML loci in Saccharomyces cerevisiae requires recruitment of Sir proteins to the HML and HMR silencers. The silencers are regulatory sites flanking both loci and consisting of binding sites for the Rap1, Abf1, and ORC proteins, each of which also functions at hundreds of sites throughout the genome in processes unrelated to silencing. Interestingly, the sequence of the binding site for Rap1 at the silencers is distinct from the genome-wide binding profile of Rap1, being a weaker match to the consensus, and indeed is bound with low affinity relative to the consensus sequence. Remarkably, this low-affinity Rap1 binding site variant was conserved among silencers of the sensu stricto Saccharomyces species, maintained as a poor match to the Rap1 genome-wide consensus sequence in all of them. We tested multiple predictions about the possible role of this binding-site variant in silencing by substituting the native Rap1 binding site at the HMR-E silencer with the genome-wide consensus sequence for Rap1. Contrary to the predictions from the current models of Rap1, we found no influence of the Rap1 binding site version on the kinetics of establishing silencing, nor on the maintenance of silencing, nor the extent of silencing. We further explored implications of these findings with regard to prevention of ectopic silencing, and deduced that the selective pressure for the unprecedented conservation of this binding site variant may not be related to silencing.National Science Foundation (U.S.) (Predoctoral Fellowship)National Institutes of Health (U.S.) (Grant GM31105

RNA-seq in the tetraploid Xenopus laevis enables genome-wide insight in a classic developmental biology model organism

Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and nonmodel organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence

The function and regulation of the GATA factor ELT-2 in the C. elegans endoderm

ELT-2 is the major regulator of genes involved in differentiation, maintenance and function o

Asymmetric Transcript Discovery by RNA-seq in C. elegans Blastomeres Identifies neg-1, a Gene Important for Anterior Morphogenesis

After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis

Multicellular organisms must have complex immune systems to detect and defeat pathogens. Plants rely on nucleotide binding site leucine rich repeat (NLR) intracellular receptors to detect pathogens. For hundreds of years, plant breeders have selected for disease-resistance traits derived from NLR genes. Despite the molecular cloning of the first NLRs more than 20 y ago, we still do not understand how these sensors function at a mechanistic level. Here, we identified a truncated NLR protein that activates cell death in response to a specific pathogen effector. Understanding how truncated NLRs function will provide a better mechanistic understanding of the plant immune system and an expanded toolkit with which to engineer disease resistance rationally in crops

Asymmetric Transcript Discovery by RNA-seq in <i>C</i>. <i>elegans</i> Blastomeres Identifies <i>neg-1</i>, a Gene Important for Anterior Morphogenesis

After fertilization but prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres, each of which goes on to generate distinct cell lineages. To understand how patterns of RNA inheritance and abundance arise after this first asymmetric cell division, we pooled hand-dissected AB and P1 blastomeres and performed RNA-seq. Our approach identified over 200 asymmetrically abundant mRNA transcripts. We confirmed symmetric or asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of the P1-enriched transcripts chs-1 and bpl-1. We screened transcripts enriched in a given blastomere for embryonic defects using RNAi. The gene neg-1 (F32D1.6) encoded an AB-enriched (anterior) transcript and was required for proper morphology of anterior tissues. In addition, analysis of the asymmetric transcripts yielded clues regarding the post-transcriptional mechanisms that control cellular mRNA abundance during asymmetric cell divisions, which are common in developing organisms.</p

Dataset associated with "mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage"

This dataset contains all primary microscopy data presented or analyzed in the publication "mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage." Source data and code used for creation of plots can be found at https://github.com/muellerflorian/parker-rna-loc-elegans.Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription. This presents an opportunity to study mechanisms of post-transcriptional regulatory control. In seeking the mechanisms behind this patterning, we discovered that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage), and P-bodies (associated with RNA processing). Transcripts differed in their dependence on 3'UTRs and RNA Binding Proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling these, we untangled a long-standing question: Are mRNAs directed to P granules for translational repression or do they accumulate there as a downstream step? We found translational repression preceded P granule localization and could occur independent of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. Overall, we show transcripts important for germline development are directed to P granules by translational repression, and this, in turn, directs their accumulation in the progenitor germ lineage where their repression can ultimately be relieved.National Institutes of Health R35GM124877.Boettcher Webb-Waring.NSF GAUSSI training grant (DGE-1450032).Some funding through Institut Pasteur

TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.This work was supported by the National Science Foundation (grant IOS-1758400 to J.L.D. and M.T.N.) and National Institutes of Health (grants GM107444 to J.L.D., RF1AG013730 to J.M., and R01NS087632 to J.M. and A.D.). J.L.D. is a Howard Hughes Medical Institute (HHMI) Investigator. M.T.N. was supported by startup funds from Colorado State University. R.G.A. was supported by an NIH Ruth L. Kirschstein NRSA fellowship (F32GM108226). K.E. was an HHMI Medical Research Fellow. F.M. is supported by a grant from the Gordon and Betty Moore Foundation (GBMF4725) to the Two Blades Foundation.Peer reviewe

Functions associated with AB and P₁-enriched transcripts.

<p><b>(A)</b> GO ontology terms over-represented in genes whose transcripts were AB-enriched at the 2-cell stage of development. Categories were identified using the GOrilla algorithm for identifying categorical significance from ranked lists [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005117#pgen.1005117.ref050" target="_blank">50</a>] and were summarized using the REViGO algorithm [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005117#pgen.1005117.ref051" target="_blank">51</a>]. <b>(B)</b> Same as A for P₁-enriched.</p