Nuclear degradation of aberrant mRNAs in Saccharomyces cerevisiae is accomplished by the nuclear exosome and its cofactors TRAMP/CTEXT. Evidence from this investigation establishes a universal role of the Nrd1p-Nab3p-Sen1p (NNS) complex in the nuclear decay of all categories of aberrant mRNAs. In agreement with this, both nrd1-1 and nrd1-2 mutations impaired the decay of all classes of aberrant messages. This phenotype is similar to that displayed by GAL::RRP41 and rrp6-Δ mutant yeast strains. Remarkably, however, nrd1ΔCID mutation (lacking the C-terminal domain required for interaction of Nrd1p with RNAPII) only diminished the decay of aberrant messages with defects occurring during the early stage of mRNP biogenesis, without affecting other messages with defects generated later in the process. Co-transcriptional recruitment of Nrd1p on the aberrant mRNAs was vital for their concomitant decay. Strikingly, this recruitment on to mRNAs defective in the early phases of biogenesis is solely dependent upon RNAPII. In contrast, Nrd1p recruitment onto export-defective transcripts with defects occurring in the later stage of biogenesis is independent of RNAPII and dependent on the CF1A component, Pcf11p, which explains the observed characteristic phenotype of nrd1ΔCID mutation. Consistently, pcf11-2 mutation displayed a selective impairment in the degradation of only the export-defective messages.
In all eukaryotes, selective nuclear degradation of aberrant mRNAs by nuclear exosome and its cofactors TRAMP, and CTEXT contribute to the fidelity of the gene expression pipeline. In the model eukaryote, Saccharomyces cerevisiae, the Nrd1p-Nab3p-Sen1p (NNS) complex, involved in the transcription termination of non-coding and several coding RNAs, was implicated in the nuclear decay of faulty messages. Consistently, nrd1-1/nrd1-2 mutant cells display impairment of the decay of all kinds of aberrant mRNAs, like the yeast mutants deficient in Rrp41p, Rrp6p, and Rrp4p. nrd1Δ CID mutation (consisting of Nrd1p lacking its CID domain thereby abrogating its interaction with RNAPII); however, abolishes the decay of aberrant messages generated during early phases of mRNP biogenesis (transcription elongation, splicing and 3′-end maturation) without affecting the decay rate of the exportdefective mRNAs. Mutation in the 3′-end processing factor, Pcf11p, in contrast, displayed a selective abolition of the decay of the aberrant mRNAs, generated at the late phase of mRNP biogenesis (exportdefective mRNAs) without influencing the faulty messages spawned in the early phase of mRNP biogenesis. Co-transcriptional recruitment of Nrd1p onto the faulty messages, which relies on RNAPII during transcription elongation and on Pcf11p post transcription, is vital for the exosomal decay of aberrant mRNAs as Nrd1p deposition to the export-defective messages found to lead to the Rrp6p recruitment followed by their decay. Thus, Nrd1p recruitment on aberrant mRNAs to make an 'Nrd1p mark' appears rate-limiting for the distinction of the aberrant messages from their normal functional counterparts.
Single-cell RNA sequencing (scRNA seq) generates highly dimensional transcriptome data to understand cellular phenotypes. Due to high cost and volume of data obtained from these experiments, most studies analyze few unique samples. Pre-analytical tissue processing and different sequencing techniques can alter quantity and phenotype of cells being examined. This study used existing human liver scRNA seq datasets to determine if heterogeneity exists between datasets and if these data could be integrated to define phenotypes across leukocyte subpopulations in healthy human liver. CD45+ cells from three published studies reporting scRNA seq data from human livers were analyzed and leukocyte subpopulations were examined. All three datasets co-clustered, but with differing cell proportions. Expression correlation demonstrated similarity across all studies. Detailed analysis of differential expression identified meta-signatures for each hepatic immune subpopulation. Herein, we introduce a novel and rigorous framework for meta-analysis of scRNA seq datasets and highlight profiles of liver immune homeostasis.
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