Protocol for generating customizable and reproducible plots of sequencing coverage data using the seqNdisplayR package.

The widespread usage of next-generation sequencing methods for functional genomics studies requires standardized tools for consistent visualization of the associated data. Here, we present seqNdisplayR, an R package for plotting standard sequencing data coverage within a genomic region of interest in a customizable and reproducible manner. We describe steps for installing software, preparing data files, choosing options, and plotting data. This tool is readily available for users with no prior experience with R through the "Shiny app" interface. For complete details on the use and execution of this protocol, please refer to Lykke-Andersen et al.,1 Gockert et al.,2 and Rouviere et al.3.

Dual agonistic and antagonistic roles of ZC3H18 provide for co-activation of distinct nuclear RNA decay pathways.

The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome is considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates.

Rapid factor depletion highlights intricacies of nucleoplasmic RNA degradation.

Turnover of nucleoplasmic transcripts by the mammalian multi-subunit RNA exosome is mediated by two adaptors: the Nuclear EXosome Targeting (NEXT) complex and the Poly(A) tail eXosome Targeting (PAXT) connection. Functional analyses of NEXT and PAXT have largely utilized long-term factor depletion strategies, facilitating the appearance of indirect phenotypes. Here, we rapidly deplete NEXT, PAXT and core exosome components, uncovering the direct consequences of their acute losses. Generally, proteome changes are sparse and largely dominated by co-depletion of other exosome and adaptor subunits, reflecting possible subcomplex compositions. While parallel high-resolution 3' end sequencing of newly synthesized RNA confirms previously established factor specificities, it concomitantly demonstrates an inflation of long-term depletion datasets by secondary effects. Most strikingly, a general intron degradation phenotype, observed in long-term NEXT depletion samples, is undetectable upon short-term depletion, which instead emphasizes NEXT targeting of snoRNA-hosting introns. Further analysis of these introns uncovers an unusual mode of core exosome-independent RNA decay. Our study highlights the accumulation of RNAs as an indirect result of long-term decay factor depletion, which we speculate is, at least partly, due to the exhaustion of alternative RNA decay pathways.

The RNA exosome shapes the expression of key protein-coding genes.

The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or components of exosome adaptor complexes, we identify ∼2900 transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts. At least 1000 of these overlap with annotated mRNA TSSs and a considerable portion of their transcripts share the annotated mRNA 3' end. We identify two types of pc-genes, both employing a single, annotated TSS across cells, but the first type primarily produces full-length, exosome-sensitive transcripts, whereas the second primarily produces prematurely terminated transcripts. Genes within the former type often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to upstream TSSs on the opposite strand. Conversely, when genes have multiple active TSSs, alternative TSSs that produce exosome-sensitive transcripts typically do not contribute substantially to overall gene expression, and most such transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-genes and imply a direct role for nuclear RNA turnover in the regulation of a subset of pc-genes.