Defining the True Native Ends of RNAs at Single-Molecule Level with TERA-Seq.

Turnover of messenger RNAs (mRNAs) is a highly regulated process and serves to control expression of RNA molecules and to eliminate aberrant transcripts. Profiling mRNA decay using short-read sequencing methods that target either the 5' or 3' ends of RNAs, overlooks valuable information about the other end, which could provide significant insights into biological aspects and mechanisms of RNA decay. Oxford Nanopore Technology (ONT) is rapidly emerging as a powerful platform for direct sequencing of native, single-RNA molecules.

TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization.

Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization.

Modulation of Aub-TDRD interactions elucidates piRNA amplification and germplasm formation.

Aub guided by piRNAs ensures genome integrity by cleaving retrotransposons, and genome propagation by trapping mRNAs to form the germplasm that instructs germ cell formation. Arginines at the N-terminus of Aub (Aub-NTRs) interact with Tudor and other Tudor domain-containing proteins (TDRDs). Aub-TDRD interactions suppress active retrotransposons via piRNA amplification and form germplasm via generation of Aub-Tudor ribonucleoproteins.

An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in .

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga , several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood.

Capturing 5' and 3' native ends of mRNAs concurrently with Akron sequencing.

Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3' or 5' terminus of RNAs, thus missing mechanistic aspects that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5' ends of uncapped, polyadenylated mRNAs and 3' ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution.

Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis.

mRNAs transmit the genetic information that dictates protein production and are a nexus for numerous pathways that regulate gene expression. The prevailing view of canonical mRNA decay is that it is mediated by deadenylation and decapping followed by exonucleolysis from the 3' and 5' ends. By developing Akron-seq, a novel approach that captures the native 3' and 5' ends of capped and polyadenylated RNAs, respectively, we show that canonical human mRNAs are subject to repeated cotranslational and ribosome-phased endonucleolytic cuts at the exit site of the mRNA ribosome channel, in a process that we term ribothrypsis.

Identification of in vivo, conserved, TAF15 RNA binding sites reveals the impact of TAF15 on the neuronal transcriptome.

RNA binding proteins (RBPs) have emerged as major causative agents of amyotrophic lateral sclerosis (ALS). To investigate the function of TAF15, an RBP recently implicated in ALS, we explored its target RNA repertoire in normal human brain and mouse neurons. Coupling high-throughput sequencing of immunoprecipitated and crosslinked RNA with RNA sequencing and TAF15 knockdowns, we identified conserved TAF15 RNA targets and assessed the impact of TAF15 on the neuronal transcriptome.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems.

A yeast functional screen predicts new candidate ALS disease genes.

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis.

RNA dysregulation in diseases of motor neurons.

Motor neuron diseases (MNDs) are neurodegenerative disorders that lead to paralysis and typically carry a dismal prognosis. In children, inherited spinal muscular atrophies are the predominant diseases that affect motor neurons, whereas in adults, amyotrophic lateral sclerosis, which is inherited but mostly sporadic, is the most common MND. In recent years, we have witnessed a revolution in this field, sparked by the discovery of the genes that cause MNDs.

Turnover of Mature miRNAs and siRNAs in Plants and Algae.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) play important roles in gene regulation and defense responses against transposons and viruses in eukaryotes. These small RNAs generally trigger the silencing of cognate sequences through a variety of mechanisms, including RNA degradation, translational inhibition and transcriptional repression. In the past few years, the synthesis and the mode of action of miRNAs and siRNAs have attracted great attention.

Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas.

Regulation of gene expression by small RNAs ( approximately 20-30 nucleotides in length) plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) commonly direct the inactivation of cognate sequences through a variety of mechanisms, including RNA degradation, translation inhibition, and transcriptional repression. Recent studies have provided considerable insight into the biogenesis and the mode of action of miRNAs and siRNAs.

Turnover of mature miRNAs and siRNAs in plants and algae.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) play important roles in gene regulation and defense responses against transposons and viruses in eukaryotes. These small RNAs generally trigger the silencing of cognate sequences through a variety of mechanisms, including RNA degradation, translational inhibition and transcriptional repression. In the past few years, the synthesis and the mode of action of miRNAs and siRNAs have attracted great attention.

Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts.

Double-stranded RNA, processed to small interfering RNAs (siRNAs) by Dicer and incorporated into the RNA-induced silencing complex (RISC), triggers gene silencing by a variety of pathways in eukaryotes. RNA interference involving the degradation of homologous transcripts is the best-characterized mechanism. However, the fate of the RNA fragments resulting from siRNA-directed cleavage is poorly understood.

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta.

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters.