Degradation Of Aberrant Transcripts Research Articles

Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway.

The 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway is an innate immune system that protects hosts against pathogenic viruses and bacteria through cleavage of exogenous single-stranded RNA; however, this system's selective targeting mechanism remains unclear. Here, we identified an mRNA quality control factor Dom34 as a novel restriction factor for a positive-sense single-stranded RNA virus. Downregulation of Dom34 and RNase L increases viral replication, as well as half-life of the viral RNA. Dom34 directly binds RNase L to form a surveillance complex to recognize and eliminate the exogenous RNA in a manner dependent on translation. Interestingly, the feature detected by the surveillance complex is not the specific sequence of the viral RNA but the ‘exogenous nature’ of the RNA. We propose the following model for the selective targeting of exogenous RNA; OAS3 activated by the exogenous RNA releases 2′-5′-oligoadenylates (2–5A), which in turn converts latent RNase L to an active dimer. This accelerates formation of the Dom34-RNase L surveillance complex, and its selective localization to the ribosome on the exogenous RNA, thereby promoting degradation of the RNA. Our findings reveal that the selective targeting of exogenous RNA in antiviral defense occurs via a mechanism similar to that in the degradation of aberrant transcripts in RNA quality control.

Abstract 649: Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes

Abstract Background: High-throughput sequencing efforts in molecular tumor diagnostics detect increased numbers of novel genetic variants, including the ones that are predicted to affect splicing. Current approach for predicting whether a specific variant may result in aberrant RNA transcripts often consists of in-silico prediction using bioinformatics prediction tools, which is sometimes followed by RT-PCR analysis of the RNA extracted from blood, or functional splicing reporter assays. Although high-quality patient RNA analysis is usually preferred, such RNA is not always available, or the analysis is hampered due to degradation of aberrant transcripts through nonsense-mediated decay. To overcome this issue, we investigated the possibility of analyzing RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues, with the underlying hypothesis that formalin fixation may inhibit RNA degradation, enabling the detection of aberrant RNA products. Methods: RNA analysis was performed for 13 canonical splice site variants predicted or known to affect splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Of 11 variants, total nucleic acid was obtained from FFPE tumor tissues. For two variants, no FFPE material was available, but EBV-transformed B-cells carrying a splice variant were cultured. Primers were designed to amplify the exon-exon boundary predicted to be affected. Housekeeping gene expression was assessed to determine presence of RNA. Results: Total nucleic acid was successfully isolated for 10 variants from eight FFPE tumor tissues and two EBV-transformed B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known splice variant, present in the B-cell line, the known aberrant splice product could only be detected after formalin fixation of the cells, suggesting that formalin fixation possibly inhibited RNA degradation. Conclusion: Aberrant splicing was successfully identified in all known splice variants and three of four variants predicted to affect splicing, of which the splice effect was previously unknown. Using a RNA sequencing approach, somatic splice variants could be easily and rapidly analyzed, enabling retrospective analysis of variants predicted to result in splicing when only FFPE material is available in clinical settings. Citation Format: Anne M. Jansen, Heleen M. van der Klift, Jaap D. van Eendenburg, Carli M. Tops, Juul T. Wijnen, Frederik J. Hes, Ajay Goel, Hans Morreau, Tom van Wezel. Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 649.

Fine Characterization of the Recurrent c.1584+18672A>G Deep-Intronic Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts

The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery.

Degradation of a Polyadenylated rRNA Maturation By-Product Involves One of the Three RRP6-Like Proteins in Arabidopsis thaliana

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

Negative selection pressure against premature protein truncation is reduced by both alternative splicing and diploidy

The importance of alternative splicing in many genomes has raised interesting questions about its role in evolution. We previously reported that alternative splicing appears to be associated with an increased rate of recent exon creation / loss events, and proposed that it can reduce negative selection pressure on sequence regions that are alternatively spliced. In this paper we test this idea directly, using the occurrence of premature protein truncation events as a metric of selection pressure. We have analyzed 13,384 full-length transcript isoforms from human and 2,227 isoforms from mouse to identify sequences containing premature termination codons (PTC) that are likely targets of mRNA nonsense mediated decay. We found that alternatively spliced isoforms indeed had a much higher frequency of PTCs (11.1%) compared with the major transcript form of each gene (3.7%). However, this effect was strongly influenced by the chromosomal location of the gene: on the X chromosome, which is generally expressed as a single copy, the overall PTC rate was much lower (3.5%, vs. 8.9% on diploid autosomes), and the effect of alternative splicing was enhanced, causing a four-fold reduction in negative selection against PTC. Thus, diploidy and alternative splicing each increased tolerance for PTC by about three-fold, as approximately additive effects. These data may suggest genomic evidence that nonsense mediated decay has itself reduced negative selection pressure during evolution, via rapid degradation of aberrant transcripts that might yield dominant negative phenotypes.