A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at Bact 29 state, an active spliceosome but not catalytically primed, which depends on SNIP1 (Smad Nuclear Interacting Protein 1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact component preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake for the spliceosome pausing, and that its misregulation contributes to neurodegeneration.

Re-polyadenylation occurs predominantly on maternal mRNA degradation intermediates during mammalian oocyte-to-embryo transition

The nascent mRNA transcribed in the nucleus is cleaved and polyadenylated before it is transported to the cytoplasm for translation. Polyadenylation can also occur in the cytoplasm for post-transcriptional regulation, especially in neurons, oocytes and early embryos. Recently, we revealed transcriptome-wide maternal mRNA cytoplasmic re-polyadenylation during the mammalian oocyte-to-embryo transition (OET). However, the mechanism of re-polyadenylation during mammalian OET, including the sites to be re-polyadenylated and the enzymes involved, is still poorly understood. Here, by analyzing the PAIso-seq1 and PAIso-seq2 poly(A) inclusive transcriptome data during OET in mice, rats, pigs, and humans, we reveal conserved re-polyadenylation of mRNA degradation intermediates. These re-polyadenylated mRNA degradation intermediates account for over half of the polyadenylated mRNA during OET in all four species. We find that mRNA degradation intermediates for re-polyadenylation are generated through Btg4-mediated deadenylation in both mouse and human. Interestingly, the poly(A) tails on the re-polyadenylated mRNA degradation intermediates are of different lengths and contain different levels of non-A residues compared to regular polyadenylation sites, suggesting specific regulation and function of these poly(A) tails in mammalian OET. Together, our findings reveal the maternal mRNA degradation intermediates as substrates for conserved cytoplasmic dominant re-polyadenylation during mammalian OET, and uncover the mechanism of production of these mRNA degradation intermediates. These findings provide new insights into mRNA post-transcriptional regulation, and a new direction for the study of mammalian OET.

Effective ribosomal RNA depletion for single-cell total RNA-seq by scDASH

A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid the cost of sequencing non-messenger transcripts, such as ribosomal RNA (rRNA), that are usually not of-interest. Hence, there are not very many protocols that enable single-cell analysis of total RNA. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) to make it suitable for depleting rRNA sequences from single-cell total RNA-seq libraries. Our analyses show that our single-cell DASH (scDASH) method can effectively deplete rRNAs from sequencing libraries with minimal off-target non-specificity. Importantly, as a result of depleting the rRNA, the rest of the transcriptome is significantly enriched for detection.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq (“cRASL-seq”), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ∼1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ∼10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

N6-methyladenosine dynamics during early vertebrate embryogenesis

Early vertebrate embryogenesis is characterized by extensive post-transcriptional regulation during the maternal-to-zygotic transition. The N6-methyladenosine (m6A) modifications on mRNA have been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with polyadenylated mRNA levels. The majority (>70%) of maternal transcripts harbor m6A, and there is a substantial increase of m6A in the polyadenylated mRNA fraction between 0 and 2 hours post fertilization. Notably, we find strong associations between m6A, cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation (ZGA). Interestingly, the relationship between m6A and translation is strongest for peaks located in the 3’UTR, but not overlapping stop codons. Sequence analyses revealed enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. After ZGA, m6A seem to diminish the effect of miR-430 mediated degradation. The reported results improve our understanding of the combinatorial code behind post-transcriptional mRNA regulation during embryonic reprogramming and early differentiation.

Herpes Simplex Virus 1 Dramatically Alters Loading and Positioning of RNA Polymerase II on Host Genes Early in Infection

ABSTRACTHerpes simplex virus 1 (HSV-1) transcription is mediated by cellular RNA polymerase II (Pol II). Recent studies investigating how Pol II transcription of host genes is altered after HSV-1 are conflicting. Chromatin immunoprecipitation sequencing (ChIP-seq) studies suggest that Pol II is almost completely removed from host genes at 4 h postinfection (hpi), while 4-thiouridine (4SU) labeling experiments show that host transcription termination is extended at 7 hpi, implying that a significant amount of Pol II remains associated with host genes in infected cells. To address this discrepancy, we used precision nuclear run-on analysis (PRO-seq) to determine the location of Pol II to single-base-pair resolution in combination with quantitative reverse transcription-PCR (qRT-PCR) analysis at 3 hpi. HSV-1 decreased Pol II on approximately two-thirds of cellular genes but increased Pol II on others. For more than 85% of genes for which transcriptional termination could be statistically assessed, Pol II was displaced to positions downstream of the normal termination zone, suggesting extensive termination defects. Pol II amounts at the promoter, promoter-proximal pause site, and gene body were also modulated in a gene-specific manner. qRT-PCR of selected RNAs showed that HSV-1-induced extension of the termination zone strongly correlated with decreased RNA and mRNA accumulation. However, HSV-1-induced increases of Pol II occupancy on genes without termination zone extension correlated with increased cytoplasmic mRNA. Functional grouping of genes with increased Pol II occupancy suggested an upregulation of exosome secretion and downregulation of apoptosis, both of which are potentially beneficial to virus production.IMPORTANCEThis study provides a map of RNA polymerase II location on host genes after infection with HSV-1 with greater detail than previous ChIP-seq studies and rectifies discrepancies between ChIP-seq data and 4SU labeling experiments with HSV-1. The data show the effects that a given change in RNA Pol II location on host genes has on the abundance of different RNA types, including nuclear, polyadenylated mRNA and cytoplasmic, polyadenylated mRNA. It gives a clearer understanding of how HSV-1 augments host transcription of some genes to provide an environment favorable to HSV-1 replication.

LNA-enhanced DNA FIT-probes for multicolour RNA imaging

A surrogate nucleobase based on 4,4'-cyanine provided up to 195-fold enhancement of red fluorescence upon complementary RNA binding. A mixture of FIT probes allowed the localization of oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by wash-free FISH and STED.

Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

ABSTRACTThrough its interaction with the 5′ translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5′-capped and 3′-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3′ GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3′ end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection.IMPORTANCETo control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.

G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA

G3BP1, a target of TDP-43, is required for normal stress granule (SG) assembly, but the functional consequences of failed SG assembly remain unknown. Here, using both transformed cell lines and primary neurons, we investigated the functional impact of this disruption in SG dynamics. While stress-induced translational repression and recruitment of key SG proteins was undisturbed, depletion of G3BP1 or its upstream regulator TDP-43 disturbed normal interactions between SGs and processing bodies (PBs). This was concomitant with decreased SG size, reduced SG–PB docking, and impaired preservation of polyadenylated mRNA. Reintroduction of G3BP1 alone was sufficient to rescue all of these phenotypes, indicating that G3BP1 is essential for normal SG–PB interactions and SG function.