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. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregationprone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. I n the future, personalized genome sequencing will become routine, empowering us to define the genetic basis of many human diseases. Currently, however, complete genome sequencing for individuals to discover rare pathogenic mutations is still too costly and time consuming. Thus, more creative approaches are needed to accelerate the discovery of disease genes. Moreover, even once genes are revealed, the need for innovative approaches to elucidate causality remains critical.ALS, also known as Lou Gehrig's disease, is a devastating adultonset neurodegenerative disease that attacks upper and lower motor neurons (1). A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2-5 y of disease onset. ALS is mostly sporadic, but ∼10% of cases are familial. Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN, VCP, and others (2). Two of these genes, TARDBP (TDP-43) and FUS/TLS (FUS) are notable because they encode related RNA-binding proteins that harbor a prion-like domain (3-6). Moreover, both of these proteins have been identified as components of pathological inclusions in neurons of patients with ALS (7-9). Indeed, an emerging concept suggested by the association of FUS and TDP-43 to ALS is that defects in RNA metabolism might contribute to disease pathogenesis. These observations suggested an intriguing possibility: Could TDP-43 and FUS be just the tip of an iceberg? In other words, could other human RNA-binding proteins with properties similar to th...

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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 spongespecific 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. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.

Regulation of gene expression by small RNAs (∼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. However, relatively little is known about mechanisms of quality control and small RNA decay in RNA interference (RNAi) pathways. Here we show that deletion of MUT68, encoding a terminal nucleotidyltransferase in the alga Chlamydomonas reinhardtii, results in elevated miRNA and siRNA levels. We found that MUT68 plays a role in the untemplated uridylation of the 3′ ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Moreover, RRP6 depletion also leads to accumulation of small RNAs in vivo. We propose that MUT68 and RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as a quality control mechanism to eliminate dysfunctional or damaged small RNA molecules.exosome | RNA interference | miRNA quality control

Messenger RNAs 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 native 3′ and 5′ ends of capped and polyadenylated RNAs respectively, we show that canonical human mRNAs are subject to repeated, cotranslational, ribosome-phased, endonucleolytic cuts at the exit site of the mRNA ribosome channel, in a process that we term ribothrypsis. We uncover RNA G-quadruplexes among likely ribothrypsis triggers, and show that ribothrypsis is a conserved process. Strikingly, we find that mRNA fragments are prevalent in living cells with important implications for the interpretation of experiments, such as RNA-Seq, that rely on the assumption that mRNAs exist largely as full-length molecules in vivo.

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. We have identified a gene ( MUT68 ) in the unicellular green alga Chlamydomonas reinhardtii that is required for the efficient decay of siRNA-targeted transcripts. MUT68 encodes a noncanonical polyadenylate polymerase that adds untemplated adenines to the 5′ RNA fragments after siRNA-mediated cleavage and appears to stimulate their exosome-dependent degradation.

Every successful cancer therapy story has Exhibit B, comprised of patients who either did not respond to the initial treatment or acquired resistance after a seemingly curative intervention. The CD19-directed chimeric antigen receptorarmed T-cell therapy (commonly known as CART-19) is the case in point. Although it has revolutionized treatment for B-cell acute lymphoblastic leukemia (B-ALL) in children and adults and gained swift FDA approval,~30% of

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. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5′ and 3′ ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5′ ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.