Abstract Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening without introducing exogenous sequence at the locus interest or marker mutations elsewhere. To this end, we describe coconversion strategy, CRISPR/Cas9 which for dominant phenotypic oligonucleotide-templated conversion event...

Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m 7 G-capped 5′ ends with high-throughput sequencing. TL-seq identified start sites for the majority yeast genes revealed many examples intragenic heterogeneity. Surprisingly, transcription initiation within 6% protein-coding regions, these were concentrated near ORFs. Furthermore, ribosome...

Cellular translation surveillance rescues ribosomes that stall on problematic mRNAs. During surveillance, endonucleolytic cleavage of the mRNA is a critical step in rescuing stalled ribosomes. Here we identify NONU-1 as factor required for pathways including no-go and nonstop decay. We show (1) reduces levels; (2) contains an Smr RNase domain decay; (3) architecture catalytic residues are conserved throughout metazoans eukaryotes, respectively; (4) formation fragments vicinity extend our...

Nonsense-mediated mRNA decay is the process by which mRNAs bearing premature stop codons are recognized and cleared from cell. While considerable information has accumulated regarding recognition of codon, less known about ensuing suppression. During characterization a second, distinct translational surveillance pathway (nonstop decay), we trapped intermediates in nonsense degradation. We present data support model wherein nonsense-mediated funnels into nonstop Caenorhabditis elegans....

Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that selection biases view transcriptome by selecting longer tailed mRNA species.Here, we show Oxford Nanopore direct RNA sequencing. As expected, skews sequenced mRNAs toward tail lengths. Interestingly, identify population...

As ribosomes translate the genetic code, they can encounter a variety of obstacles that hinder their progress. If stall for prolonged times, cells suffer due to loss translating and accumulation aberrant protein products. Thus protect cells, stalled experience series reactions relieve degrade offending mRNA, process known as No-Go mRNA Decay (NGD). While much machinery NGD is known, precise ordering events factors along this pathway has not been tested. Here, we deploy C . elegans unravel...

In the course of identifying and cleaving RNA, RNAi machinery must encounter contend with megadalton-sized ribosomes that carry out translation. We investigated this interface by examining fate actively translated mRNAs subjected to in C. elegans Quantifying RNA levels (RNA-seq) ongoing translation (Ribo-seq), we found there is a greater fold repression than expected from loss alone, observing stronger relative for multiple, independent double-stranded triggers, multiple genes. animals lack...

Abstract Transposons can impact the host genome by altering gene expression and participating in chromosome rearrangements. Therefore, organisms evolved different ways to minimize level of transposition. In Saccharomyces cerevisiae its close relative S. paradoxus, Ty1 copy number control (CNC) is mediated self-encoded restriction factor p22, which derived from GAG capsid inhibits virus-like particle (VLP) assembly function. Based on secondary screens cofactors, we identified LOC1, a RNA...

Nonsense-mediated mRNA decay (NMD) protects cells from the toxic and potentially dominant effects of truncated proteins. Targeting mRNAs with early stop codons is mediated by ribosome spatiotemporally aligned translation termination. Previously we identified a novel NMD intermediate: ribosomes stalled on cleaved codons, raising possibility that begins even prior to removal codon. Here show this intermediate result cleavage endonuclease SMG-6. Our work supports model in which stall secondary...

ABSTRACT Cellular translation surveillance rescues ribosomes that stall on problematic mRNAs. During surveillance, endonucleolytic cleavage of the mRNA is a critical step in rescuing stalled ribosomes. However, nuclease(s) responsible remain unknown. Here we identify NONU-1 as novel endoribonuclease required for pathways including No-Go and Nonstop Decay. We show that: (1) reduces levels; (2) contains an Smr RNase domain decay with properties similar to unknown endonuclease; (3) architecture...

Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation strand-specific small pools from bacteria that can be multiplexed to accommodate multiple biological samples single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis treated with T4 polynucleotide kinase. This allows 3' adapter ligation CRISPR RNAs, which don't have pre-existing 3'-OH ends. Pre-adenylated...

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression tissue patterning in animals. An invaluable tool modern research presence a high-resolution ribosome structure, though no such structure exists C. . Here, we present single-particle cryogenic electron microscopy (cryo-EM) reconstruction molecular ribosome, revealing significantly streamlined animal ribosome. Many facets are conserved ,...

ABSTRACT Premature stop codon-containing mRNAs can produce truncated and dominantly acting proteins that harm cells. Eukaryotic cells protect themselves by degrading such via the Nonsense-Mediated mRNA Decay (NMD) pathway. The precise reactions which attack NMD target remain obscure, precluding a mechanistic understanding of hampering therapeutic efforts to control NMD. A key step in is decay mRNA, proposed occur several competing models including deadenylation, exonucleolytic decay, and/or...