The multiple functions of RNA helicases as drivers and regulators of gene expression.

RNA helicases comprise the largest family of enzymes involved in the metabolism of mRNAs, the processing and fate of which rely on their packaging into messenger ribonucleoprotein particles (mRNPs). In this Review, we describe how the capacity of some RNA helicases to either remodel or lock the composition of mRNP complexes underlies their pleiotropic functions at different steps of the gene expression process. We illustrate the roles of RNA helicases in coordinating gene expression steps and programmes, and propose that RNA helicases function as molecular drivers and guides of the progression of their mRNA substrates from one RNA-processing factory to another, to a productive mRNA pool that leads to protein synthesis or to unproductive mRNA pools that are stored or degraded.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

SARS-CoV-2 NSP13 Inhibits Type I IFN Production by Degradation of TBK1 via p62-Dependent Selective Autophagy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.

BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.

The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

Role of stress granules in modulating senescence and promoting cancer progression: Special emphasis on glioma.

Stress granules (SGs) contain mRNAs and proteins stalled in translation during stress; these are increasingly being implicated in diseases, including neurological disorders and cancer. The dysregulated assembly, persistence, disassembly and clearance of SGs contribute to the process of senescence. Senescence has long been a mysterious player in cellular physiology and associated diseases. The systemic process of aging has been pivotal in the development of various neurological disorders like age-related neuropathy, Alzheimer's disease and Parkinson's disease. Glioma is a cancer of neurological origin with a very poor prognosis and high rate of recurrence, SGs have only recently been implicated in its pathogenesis. Senescence has long been established to play an antitumorigenic role, however, relatively less studied is its protumorigenic importance. Here, we have evaluated the existing literature to assess the crosstalk of the two biological phenomena of senescence and SG formation in the context of tumorigenesis. In this review, we have attempted to analyze the contribution of senescence in regulating diverse cellular processes, like, senescence associated secretory phenotype (SASP), microtubular reorganization, telomeric alteration, autophagic clearance and how intricately these phenomena are tied with the formation of SGs. Finally, we propose that interplay between senescence, its contributing factors and the genesis of SGs can drive tumorigenicity of gliomas, which can potentially be utilized for therapeutic intervention.

The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly.

Cytoplasmic pattern recognition receptors detect non-self RNAs during virus infections and initiate antiviral signaling. One receptor, MDA5, possesses essential signaling domains, but weak RNA binding. A second receptor, LGP2, rapidly detects diverse dsRNA species, but lacks signaling domains. Accumulating evidence suggests LGP2 and MDA5 work together to detect viral RNA and generate a complete antiviral response, but the basis for their cooperation has been elusive. Experiments presented here address this gap in antiviral signaling, revealing that LGP2 assists MDA5-RNA interactions leading to enhanced MDA5-mediated antiviral signaling. LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly, resulting in the formation of more numerous, shorter MDA5 filaments that are shown to generate equivalent or greater signaling activity in vivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 coactivation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses.

The role of YTH domain containing 2 in epigenetic modification and immune infiltration of pan-cancer.

YTH domain containing 2 (YTHDC2) is the largest N6-Methyladenosine (m6 A) binding protein of the YTH protein family and the only member containing ATP-dependent RNA helicase activity. For further analysing its biological role in epigenetic modification, we comprehensively explored YTHDC2 from gene expression, genetic alteration, protein-protein interaction (PPI) network, immune infiltration, diagnostic value and prognostic value in pan-cancer, using a series of databases and bioinformatic tools. We found that YTHDC2 with Missense mutation could cause a different prognosis in uterine corpus endometrial carcinoma (UCEC), and its different methylation level could lead to a totally various prognosis in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), lung squamous cell carcinoma (LUSC) and UCEC. The main molecular mechanisms of YTHDC2 focused on catalytic activity, helicase activity, snRNA binding, spliceosome and mRNA surveillance. Additionally, YTHDC2 was notably correlated with tumour immune infiltration. Moreover, YTHDC2 had a high diagnostic value for seven cancer types and a prognostic value for brain lower grade glioma (LGG), rectum adenocarcinoma (READ) and skin cutaneous melanoma (SKCM). Collectively, YTHDC2 plays a significant role in epigenetic modification and immune infiltration and maybe a potential biomarker for diagnosis and prognosis in certain cancers.

Kaposi's sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment.

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

Specification of Drosophila neuropeptidergic neurons by the splicing component brr2.

During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. In this study, we focus on the role of RNA processing during target-derived signaling. We found that brr2 and Prp8 play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.

DHX29 functions as an RNA co-sensor for MDA5-mediated EMCV-specific antiviral immunity.

Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is an RNA helicase required for the translation of 5' structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5' structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity.

Targeting RNA helicases in cancer: The translation trap.

Cancer cells are reliant on the cellular translational machinery for both global elevation of protein synthesis and the translation of specific mRNAs that promote tumor cell survival. Targeting translational control in cancer is therefore increasingly recognized as a promising therapeutic strategy. In this regard, DEAD/H box RNA helicases are a very interesting group of proteins, with several family members regulating mRNA translation in cancer cells. In this review, we delineate the mechanisms by which DEAD/H box proteins modulate oncogenic translation and how inhibition of these RNA helicases can be exploited for anti-cancer therapeutics.

Dynamic protein-protein interaction wiring of the human spliceosome.

More than 200 proteins copurify with spliceosomes, the compositionally dynamic RNPs catalyzing pre-mRNA splicing. To better understand protein - protein interactions governing splicing, we systematically investigated interactions between human spliceosomal proteins. A comprehensive Y2H interaction matrix screen generated a protein interaction map comprising 632 interactions between 196 proteins. Among these, 242 interactions were found between spliceosomal core proteins and largely validated by coimmunoprecipitation. To reveal dynamic changes in protein interactions, we integrated spliceosomal complex purification information with our interaction data and performed link clustering. These data, together with interaction competition experiments, suggest that during step 1 of splicing, hPRP8 interactions with SF3b proteins are replaced by hSLU7, positioning this second step factor close to the active site, and that the DEAH-box helicases hPRP2 and hPRP16 cooperate through ordered interactions with GPKOW. Our data provide extensive information about the spliceosomal protein interaction network and its dynamics.

mRNAs are sorted for export or degradation before passing through nuclear speckles.

A significant fraction of mRNAs are degraded by the nuclear exosome in normal cells. Here, we studied where and when these exosome target mRNAs are sorted away from properly exported ones in the cells. We show that upon exosome inactivation, polyA RNAs are apparently accumulated in nuclear foci that are distinct from nuclear speckles (NSs), and provide several lines of evidence supporting that these polyA RNAs mainly correspond to accumulating exosome target mRNAs. These results suggest that exosomal mRNA degradation mostly occurs outside of NSs. In support of this possibility, targeting exosome target mRNAs to NSs stabilizes them by preventing exosomal degradation. Furthermore, inhibiting mRNA release from NSs does not attenuate exosomal degradation in normal cells, and results in polyA RNA accumulation both inside and outside of NSs in exosome inactivated cells, suggesting that passage through NSs is not required for sorting mRNAs for degradation or export. Indeed, exosome target mRNAs that normally do not enter NSs are exported upon exosome inactivation. Together, our data suggest that exosome target mRNAs are mainly degraded in the nucleoplasm before entering NSs and rapid removal of these mRNAs is important for preventing their nuclear export.

LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.

Here we show that laboratory of genetics and physiology 2 (LGP2) virus sensor protein regulates gene expression network of endogenous genes mediated by TAR-RNA binding protein (TRBP)-bound microRNAs (miRNAs). TRBP is an enhancer of RNA silencing, and functions to recruit precursor-miRNAs (pre-miRNAs) to Dicer that processes pre-miRNA into mature miRNA. Viral infection activates the antiviral innate immune response in mammalian cells. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and LGP2, function as cytoplasmic virus sensor proteins during viral infection. RIG-I and MDA5 can distinguish between different types of RNA viruses to produce antiviral cytokines, including type I interferon. However, the role of LGP2 is controversial. We found that LGP2 bound to the double-stranded RNA binding sites of TRBP, resulting in inhibition of pre-miRNA binding and recruitment by TRBP. Furthermore, although it is unclear whether TRBP binds to specific pre-miRNA, we found that TRBP bound to particular pre-miRNAs with common structural characteristics. Thus, LGP2 represses specific miRNA activities by interacting with TRBP, resulting in selective regulation of target genes. Our findings show that a novel function of LGP2 is to modulate RNA silencing, indicating the crosstalk between RNA silencing and RLR signaling in mammalian cells.

Combined deficiency of Senataxin and DNA-PKcs causes DNA damage accumulation and neurodegeneration in spinal muscular atrophy.

Chronic low levels of survival motor neuron (SMN) protein cause spinal muscular atrophy (SMA). SMN is ubiquitously expressed, but the mechanisms underlying predominant neuron degeneration in SMA are poorly understood. We report that chronic low levels of SMN cause Senataxin (SETX)-deficiency, which results in increased RNA-DNA hybrids (R-loops) and DNA double-strand breaks (DSBs), and deficiency of DNA-activated protein kinase-catalytic subunit (DNA-PKcs), which impairs DSB repair. Consequently, DNA damage accumulates in patient cells, SMA mice neurons and patient spinal cord tissues. In dividing cells, DSBs are repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, but neurons predominantly use NHEJ, which relies on DNA-PKcs activity. In SMA dividing cells, HR repairs DSBs and supports cellular proliferation. In SMA neurons, DNA-PKcs-deficiency causes defects in NHEJ-mediated repair leading to DNA damage accumulation and neurodegeneration. Restoration of SMN levels rescues SETX and DNA-PKcs deficiencies and DSB accumulation in SMA neurons and patient cells. Moreover, SETX overexpression in SMA neurons reduces R-loops and DNA damage, and rescues neurodegeneration. Our findings identify combined deficiency of SETX and DNA-PKcs stemming downstream of SMN as an underlying cause of DSBs accumulation, genomic instability and neurodegeneration in SMA and suggest SETX as a potential therapeutic target for SMA.

The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis.

The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.

Vaccine adjuvants: putting innate immunity to work.

Adjuvants enhance immunity to vaccines and experimental antigens by a variety of mechanisms. In the past decade, many receptors and signaling pathways in the innate immune system have been defined and these innate responses strongly influence the adaptive immune response. The focus of this review is to delineate the innate mechanisms by which adjuvants mediate their effects. We highlight how adjuvants can be used to influence the magnitude and alter the quality of the adaptive response in order to provide maximum protection against specific pathogens. Despite the impressive success of currently approved adjuvants for generating immunity to viral and bacterial infections, there remains a need for improved adjuvants that enhance protective antibody responses, especially in populations that respond poorly to current vaccines. However, the larger challenge is to develop vaccines that generate strong T cell immunity with purified or recombinant vaccine antigens.

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features.

Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in Saccharomyces cerevisiae. However, Prp16 functions are largely unexplored in Schizosaccharomyces pombe, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5' splice site (5'SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5'SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the spprp16F528S mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5'SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in non-coding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in spprp16F528S cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site-snRNA interaction strength.

Yeast Sen1 helicase protects the genome from transcription-associated instability.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.

Interaction profiling identifies the human nuclear exosome targeting complex.

The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.