Essential RNA Research Articles

Characterization of the aberrant splicing of MAP3K7 induced by cancer-associated SF3B1 mutation.

SF3B1, an essential RNA splicing factor, is frequently mutated in various types of cancers, and the cancer-associated SF3B1 mutation causes aberrant RNA splicing. The aberrant splicing of several transcripts, including MAP3K7, promotes tumorigenesis. Here, we identify a premature termination codon in the aberrantly spliced transcript of MAP3K7. Treatment of HEK293T cells transfected with the K700E-mutated SF3B1 with cycloheximide leads to increased accumulation of the aberrant spliced transcript of MAP3K7, demonstrating that the aberrantly spliced transcript of MAP3K7 is targeted by nonsense-mediated decay. The aberrantly spliced MAP3K7 transcript uses an aberrant 3' splice sites and an alternative branchpoint sequence. In addition, the aberrant splicing of MAP3K7 requires not only the polypyrimidine tract associated with normal splicing but also an alternative polypyrimidine tract upstream of the aberrant 3' splice site. Other cancer-associated SF3B1 mutations also cause the aberrant splicing of MAP3K7, which depends on the same sequence features. Our data provide a further understanding of the mechanisms underlying aberrant splicing induced by cancer-associated SF3B1 mutation, and reveal an important role of alternative polypyrimidine tract in diseases.

Identification of m6A-Associated RNA Binding Proteins Using an Integrative Computational Framework.

N6-methyladenosine (m6A) is an abundant modification on mRNA that plays an important role in regulating essential RNA activities. Several wet lab studies have identified some RNA binding proteins (RBPs) that are related to m6A's regulation. The objective of this study was to identify potential m6A-associated RBPs using an integrative computational framework. The framework was composed of an enrichment analysis and a classification model. Utilizing RBPs' binding data, we analyzed reproducible m6A regions from independent studies using this framework. The enrichment analysis identified known m6A-associated RBPs including YTH domain-containing proteins; it also identified RBM3 as a potential m6A-associated RBP for mouse. Furthermore, a significant correlation for the identified m6A-associated RBPs is observed at the protein expression level rather than the gene expression level. On the other hand, a Random Forest classification model was built for the reproducible m6A regions using RBPs' binding data. The RBP-based predictor demonstrated not only competitive performance when compared with sequence-based predictions but also reflected m6A's action of repelling against RBPs, which suggested that our framework can infer interaction between m6A and m6A-associated RBPs beyond sequence level when utilizing RBPs' binding data. In conclusion, we designed an integrative computational framework for the identification of known and potential m6A-associated RBPs. We hope the analysis will provide more insights on the studies of m6A and RNA modifications.

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major.

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

POLR3-related leukodystrophy: How do mutations affecting RNA polymerase III subunits cause hypomyelination?

Hypomyelinating leukodystrophies are a group of genetic disorders characterized by insufficient myelin deposition during development. A subset of hypomyelinating leukodystrophies, named RNA polymerase III (Pol III or POLR3)-related leukodystrophy or 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) leukodystrophy, was found to be caused by biallelic variants in genes encoding subunits of the enzyme Pol III, including POLR3A, POLR3B, POLR3K, and POLR1C. Pol III is one of the three nuclear RNA polymerases that synthesizes small non-coding RNAs, such as tRNAs, 5S RNA, and others, that are involved in the regulation of essential cellular processes, including transcription, translation and RNA maturation. Affinity purification coupled with mass spectrometry (AP-MS) revealed that a number of mutations causing POLR3-related leukodystrophy impair normal assembly or biogenesis of Pol III, often causing a retention of the unassembled subunits in the cytoplasm. Even though these proteomic studies have helped to understand the molecular defects associated with leukodystrophy, how these mutations cause hypomyelination has yet to be defined. In this review we propose two main hypotheses to explain how mutations affecting Pol III subunits can cause hypomyelination.

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation.

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.

The RNA Architecture of the SARS-CoV-2 3'-Untranslated Region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

A novel essential small RNA, sSp_p6 influences nitrogen fixation in Azospirillum brasilense

Azospirillum brasilense Sp245, a diazotrophic plant growth-promoting bacterium influences host plants via well-known mechanisms, gene interactions and confers abiotic and biotic stress tolerance mediated by signalling molecules. We present here, the functional characterization of a small RNA (sRNA), sSp_p6, identified from the 468 sRNAs identified in A. brasilense Sp245 and confirmed its involvement in nitrogen metabolism in the strain. sSp_p6 expression was found to be modulated in carbon and nitrogen stress while forming an interactive network with the target genes, vnfG (encoding vanadium-dinitrogenase) and σ-54 (sigma factor, interaction region) thereby establishing an essential role of this sRNA in biological nitrogen fixation by the strain. Additionally, upon inoculation with sSp_p6 overexpressing mutant, the host plant Sorghum exhibited increased lateral root formation, which led to improved plant root morphology.

Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a widespread viral disease that has led to huge economic losses for the global swine industry. Non-structural protein 9 (Nsp9) of PRRSV possesses essential RNA-dependent RNA polymerase (RdRp) activity for viral RNA replication. Our previous report showed that Nsp9-specific nanobody, Nb6, was able to inhibit PRRSV replication. In this study, recombinant Nsp9 and Nsp9-Nb6 complex were prepared then characterized using bio-layer interferometry (BLI) and dynamic light scattering (DLS) analyses that demonstrated high-affinity binding of Nb6 to Nsp9 to form a homogeneous complex. Small-angle X-ray scattering (SAXS) characterization analyses revealed that spatial interactions differed between Nsp9 and Nsp9-Nb6 complex molecular envelopes. Enzyme-linked immunosorbent assays (ELISAs) revealed key involvement of Nsp9 residues Ile588, Asp590, and Leu643 and Nb6 residues Tyr62, Trp105, and Pro107 in the Nsp9-Nb6 interaction. After reverse genetics-based techniques were employed to generate recombinant Nsp9 mutant viruses, virus replication efficiencies were assessed in MARC-145 cells. The results revealed impaired viral replication of recombinant viruses bearing I588A and L643A mutations as compared with replication of wild type virus, as evidenced by reduced negative-strand genomic RNA [(−) gRNA] synthesis and attenuated viral infection. Moreover, the isoleucine at position 588 of Nsp9 was conserved across PRRSV genotypes. In conclusion, structural analysis of the Nsp9-Nb6 complex revealed novel amino acid interactions involved in viral RNA replication that will be useful for guiding development of structure-based anti-PRRSV agents.

PRMT5 Inhibition Affects RNA Methylation and Is a Potential Therapeutic Target in BPDCN

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and clinically aggressive hematologic cancer that arises from malignant transformation of plasmacytoid dendritic cells (pDCs) and is predisposed to leukemic transformation. Apart from the recently approved therapy, Tagraxofusp-erzs, treatment options for BPDCN remain limited. A recurrent feature of BPDCN is dysregulated MYC expression, which is associated with dependence on Protein arginine methyltransferase 5 (PRMT5). PRMT5 catalyses symmetric dimethylation of arginine residues post translationally and is often overexpressed in hematological malignancies. It was recently reported that BPDCN patient samples were enriched for a PRMT5 gene signature. However, the role of elevated PRMT5 in BPDCN has not been studied. Moreover, how pharmacological inhibition of PRMT5 affects BPDCN remains unexplored. Here, we pursue the hypothesis that PRMT5 or PRMT5-regulated factors represent a potential therapeutic opportunity in BPDCN, a hematologic malignancy of unmet clinical need. We report that elevated PRMT5 activity is critical for BPDCN. Compared to pDCs from healthy individuals, BPDCN cells expressed much higher levels of MYC and PRMT5. Functionally, in vitro growth of BPDCN cells treated with the PRMT5 inhibitor GSK3326595 (GSK595) was impaired in a dose-dependent manner, with net negative cell growth observed 6 days post-treatment. Similarly, bi-daily oral administration of GSK595 to CAL-1 xenografts significantly mitigated the growth of tumors, without any observed impact on body weight. In addition, the sensitivity of BPDCN cells to GSK595 was comparable to MOLM-13, which has been reported to be highly sensitive to PRMT5 inhibition. Correspondingly, this growth inhibition was associated with the induction of apoptosis. Thus, confirming the requirement for elevated PRMT5 levels in BPDCN. We then transcriptionally profiled GSK595-treated BPDCN cells and identified differential splicing events (DSEs) across several splicing classes; of which intron retention (and to a lesser extent, exon skipping) events were substantially elevated. Interestingly, these upregulated intron retention events were over-represented for processes associated with RNA modification. Specifically, we found that splicing of several essential genes involved in RNA modification were dysregulated. Among these genes was Methyltransferase-like protein 3 (METTL3), an RNA methyltransferase writer which mediates m6A modification on mRNAs. In agreement, we show that PRMT5 inhibition decreased the expression of METTL3 along with its downstream targets - MYC & TAZ. We also show that the levels of m6A modification in GSK595-treated BPDCN cells decreased in a dose-dependent manner; thus, highlighting the reduced functional activity of METTL3 following PRMT5 inhibition. Taken together, our data implicates METTL3 and possibly other essential RNA modification genes as key downstream effectors of PRMT5 in BPDCN. Collectively, our study highlights a novel link between PRMT5 and RNA modification in BPDCN. In addition, we show that elevated PRMT5 is important in BPDCN and its inhibition mitigates BPDCN progression. Therefore, PRMT5 inhibition is a potential therapeutic strategy for BPDCN. Disclosures No relevant conflicts of interest to declare.

SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies.

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.

Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis.

Simple SummaryRNA interference is a gene suppression tool that uses double-stranded RNA to prevent specific genes from producing proteins. By targeting essential genes RNA interference can be developed for control of insect pests. Unfortunately, RNA interference is not equally effective for all insects. Previous investigation suggested that RNA is rapidly digested by unidentified components of body fluids in the European corn borer caterpillar. We characterized genes encoding proteins from European corn borer that are associated with RNA digestion in other insects. Our results suggest that two proteins (RNA interference efficiency-related nuclease and double-stranded RNA-degrading endonuclease 2) may be responsible for digesting RNAs in the European corn borer gut, whereas two other proteins (double-stranded RNA-degrading endonuclease 1 and double-stranded RNA-degrading endonuclease 4) may be responsible for digesting RNA in European corn borer body fluid. These findings suggest digestion of RNA in the European corn borer is likely due to the activity of these proteins. These findings provide information about the mechanism(s) influencing RNA stability in insects. The knowledge generated by this study will facilitate the development of strategies for enhancing RNA interference in insects.Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

Folding heterogeneity in the essential human telomerase RNA three-way junction.

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. To compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (TWJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, secondary structural modeling, and single-molecule structural analysis. Our data suggest the essential P6.1 stem–loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. Finally, single-molecule FRET measurements of CR4/5 and a mutant designed to stabilize the P6.1 stem demonstrate that TERT binding selects for a structural conformation of CR4/5 that is not the dominant state of the TERT-free in vitro RNA ensemble.

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder.

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the ADAT3 locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.

A Chemoenzymatic Synthesis of the (RP)-Isomer of the Antiviral Prodrug Remdesivir.

The COVID-19 pandemic threatens to overwhelm healthcare systems around the world. The only current FDA-approved treatment, which directly targets the virus, is the ProTide prodrug remdesivir. In its activated form, remdesivir prevents viral replication by inhibiting the essential RNA-dependent RNA polymerase. Like other ProTide prodrugs, remdesivir contains a chiral phosphorus center. The initial selection of the (SP)-diastereomer for remdesivir was reportedly due to the difficulty in producing the pure (RP)-diastereomer of the required precursor. However, the two currently known enzymes responsible for the initial activation step of remdesivir are each stereoselective and show differential tissue distribution. Given the ability of the COVID-19 virus to infect a wide array of tissue types, inclusion of the (RP)-diastereomer may be of clinical significance. To help overcome the challenge of obtaining the pure (RP)-diastereomer of remdesivir, we have developed a novel chemoenzymatic strategy that utilizes a stereoselective variant of the phosphotriesterase from Pseudomonas diminuta to enable the facile isolation of the pure (RP)-diastereomer of the chiral precursor for the chemical synthesis of the (RP)-diastereomer of remdesivir.

MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB)

BackgroundThe rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P’s sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date.ResultsOf the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation.ConclusionsThe coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Contributions of Gene Modules Regulated by Essential Noncoding RNA in Colon Adenocarcinoma Progression.

Noncoding RNAs (ncRNAs), especially microRNA (miRNA) and long noncoding RNA (lncRNA), have an impact on a variety of important biological processes during colon adenocarcinoma (COAD) progression. This includes chromatin organization, transcriptional and posttranscriptional regulation, and cell-cell signaling. The aim of this study is to identify the ncRNA-regulated modules that accompany the progression of COAD and to analyze their mechanisms, in order to screen the potential prognostic biomarkers for COAD. An integrative molecular analysis was carried out to identify the crosstalks of gene modules between different COAD stages, as well as the essential ncRNAs in the posttranscriptional regulation of these modules. 31 ncRNA regulatory modules were found to be significantly associated with overall survival in COAD patients. 17 out of the 31 modules (in which ncRNAs played essential roles) had improved the predictive ability for COAD patient survival compared to only the mRNAs of those modules, which were enriched in the core cancer hallmark pathways with closer interactions. These suggest that the ncRNAs' regulatory modules not only exhibit close relation to COAD progression but also reflect the dynamic significant crosstalk of genes in the modules to the different malignant extent of COAD.

Indirect Bacterial Transcription-Translation Coupling Mechanism Revealed by In Situ Integrative Structural Biology

In situ structural biology aims to resolve macromolecular structures and collective behaviors of macromolecular machineries within the cell. In bacteria, transcription mediated by RNA polymerase is known to be functionally coupled to the leading ribosome, a process crucial for efficient gene expression. Direct ribosome-RNA polymerase interaction has been demonstrated in vitro, but how these two gigantic molecular machines coordinate is yet unclear, especially within the context of crowded and complex cellular environments. Using Mycoplasma pneumonia as model system, we integrated cellular cryo-electron tomography followed by sub-tomogram analysis (STA) and in-cell crosslinking mass spectrometry (CLMS) to investigate the coupling in native cells, without resorting to labelling or cell disruption. By averaging sub-tomograms extracted in silico from cellular volumes, we solved the 70S ribosome structure at 6.4 Å resolution, so far the highest resolution reported for in-cell cryo-ET. After large-scale classification, we determined the structure of a super-complex consisting of ribosome, RNA polymerase and other auxiliary proteins. Protein-protein interaction networks revealed by in-cell CLMS indicated an essential transcription elongation factor bridging RNA polymerase and the translating ribosome. Integrative modelling suggests a novel, indirect transcription-translation coupling mechanism, which advances our understanding of key machineries of the central dogma of molecular biology in bacteria. Methodology wise, our work demonstrates the feasibility and superiority of integrative structural biology performed directly inside cells. Integration of two approaches, cryo-ET/STA and in-cell CLMS, holds great potential to delineate cellular processes in a quantitative and structural view.

From parts lists to functional significance-RNA-protein interactions in gene regulation.

Hundreds of canonical RNA binding proteins facilitate diverse and essential RNA processing steps in cells forming a central regulatory point in gene expression. However, recent discoveries including the identification of a large number of noncanonical proteins bound to RNA have changed our view on RNA-protein interactions merely as necessary steps in RNA biogenesis. As the list of proteins interacting with RNA has expanded, so has the scope of regulation through RNA-protein interactions. In addition to facilitating RNA metabolism, RNA binding proteins help to form subcellular structures and membraneless organelles, and provide means to recruit components of macromolecular complexes to their sites of action. Moreover, RNA-protein interactions are not static in cells but the ribonucleoprotein (RNP) complexes are highly dynamic in response to cellular cues. The identification of novel proteins in complex with RNA and ways cells use these interactions to control cellular functions continues to broaden the scope of RNA regulation in cells and the current challenge is to move from cataloguing the components of RNPs into assigning them functions. This will not only facilitate our understanding of cellular homeostasis but may bring in key insights into human disease conditions where RNP components play a central role. This review brings together the classical view of regulation accomplished through RNA-protein interactions with the novel insights gained from the identification of RNA binding interactomes. We discuss the challenges in combining molecular mechanism with cellular functions on the journey towards a comprehensive understanding of the regulatory functions of RNA-protein interactions in cells. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications aRNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition.

Functional Loss of ATRX and TERC Activates Alternative Lengthening of Telomeres (ALT) in LAPC4 Prostate Cancer Cells.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. IMPLICATIONS: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.