Biology subjects

Blencowe, B.

Publications and source records attributed to Blencowe, B..

6 recordsLinked to original sources

bioRxiv2021

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

molecular biology↗

bioRxiv2021

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types

systems biology↗

bioRxiv2020

SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs

The COVID-19 pandemic has caused over one million deaths thus far. There is an urgent need for the development of specific viral therapeutics and a vaccine. SARS-CoV-2 nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for both antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we initially catalogued the protein-protein interactions of 21 SARS-CoV-2 proteins in HEK293 cells, finding that the stress granule resident proteins G3BP1 and G3BP2 copurify with N with high specificity. We demonstrate that N protein expression in human cells sequesters G3BP1 and G3BP2 through its physical interaction with these proteins, attenuating stress granule (SG) formation. The ectopic expression of G3BP1 in N-expressing cells was sufficient to reverse this phenotype. Since N is an RNA-binding protein, we performed iCLIP-sequencing experiments in cells, with or without exposure to oxidative stress, to identify the host RNAs targeted by N. Our results indicate that SARS-CoV-2 N protein binds directly to thousands of mRNAs under both conditions. Like the G3BPs stress granule proteins, N was found to predominantly bind its target mRNAs in their 3UTRs. RNA sequencing experiments indicated that expression of N results in wide-spread gene expression changes in both unstressed and oxidatively stressed cells. We suggest that N regulates host gene expression by both attenuating stress granules and binding directly to target mRNAs.

molecular biology↗

bioRxiv2020

Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA transcripts is driven by intron retention: implications for RNA-directed therapies

Numerous global connections have been made between splicing and other layers of gene regulation, including the spatial partitioning of the transcriptome in the cell. Yet, there has been surprisingly little analysis of the spatio-temporal regulation of individual protein-coding and non-coding RNA molecules in single cells. Here we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH revealed that nuclear TERT transcripts uniformly and robustly retain two specific introns whose splicing occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution had significant effects on cell growth. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of RNAs within the cell; this process reveals new targets for RNA-based therapies.

cell biology↗

bioRxiv2019

Regulation of transcription termination by FUS and TDP-43

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. We previously found that symmetrical dimethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin. SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS). R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia). Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway. Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.

molecular biology↗

bioRxiv2019

Ubiquitylated H2A.Z nucleosomes are associated with nuclear architectural proteins and global transcriptional silencing

H2A.Z monoubiquitylation has been linked to transcriptional repression, but the molecular details involved remain unclear. To address this, we developed a unique ubiquitylation-dependent auto-biotinylation approach, named H2A.Z-UAB, to purify ubiquitylated H2A.Z (H2A.Zub) mononucleosomes for biochemical and genome-wide analyses. We found that H2A.Zub nucleosomes are enriched for the repressive histone post-translational modification H3K27me3 but depleted of H3K4 methylation and other active transcription-associated modifications. ChIP-seq analyses revealed that H2A.Zub-nucleosomes associate with repressive chromatin states and are mostly enriched at genes with low or no expression. Consistent with the biochemical analyses, H2A.Zub ChIP-seq peaks align with regions depleted of H3K4 methylation, H3K27 acetylation, but enriched for H3K27me3. We further observed a strong correlation between high levels of H2A.Zub and DNA methylation, particularly at methylated CpG-rich islands. Finally, we found that a significant proportion of H2A.Zub peaks correspond to homopurine/homopyrimidine sequences that are generally linked to silenced genes and repressive chromatin. Collectively, these findings suggest that H2A.Z ubiquitylation plays an overarching role in global transcriptional silencing through its association with multiple types of repressive mechanisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/759852v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@71804eorg.highwire.dtl.DTLVardef@19eb987org.highwire.dtl.DTLVardef@1a67e15org.highwire.dtl.DTLVardef@19fc5c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗