In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance, however, ...it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hnRNP particles determines their effect on the inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens ...of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3′ end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals.
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•Multivalent binding sites in young LINEs mainly recruit repressive RBPs•PTBP1 and MATR3 co-bind and repress RNA processing within and around young LINEs•1-kb exclusion zone of LINEs from vicinity of exons reflects their repressive effects•Sequence-driven RBP remodeling on old LINEs drives evolution of tissue-specific exons
LINE elements recruit RNA-binding proteins to mammalian introns, influencing splicing and playing a key role in the evolution of tissue-specific exons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Astrocytes contribute to motor neuron death in amyotrophic lateral sclerosis (ALS), but whether they adopt deleterious features consistent with inflammatory reactive states remains incompletely ...resolved. To identify inflammatory reactive features in ALS human induced pluripotent stem cell (hiPSC)-derived astrocytes, we examined transcriptomics, proteomics, and glutamate uptake in
-mutant astrocytes. We complemented this by examining other ALS mutations and models using a systematic meta-analysis of all publicly-available ALS astrocyte sequencing data, which included hiPSC-derived astrocytes carrying
,
, and
gene mutations as well as mouse ALS astrocyte models with
mutation,
deletion, and
(also known as membralin) deletion. ALS astrocytes were characterized by up-regulation of genes involved in the extracellular matrix, endoplasmic reticulum stress, and the immune response and down-regulation of synaptic integrity, glutamate uptake, and other neuronal support processes. We identify activation of the TGFB, Wnt, and hypoxia signaling pathways in both hiPSC and mouse ALS astrocytes. ALS changes positively correlate with TNF, IL1A, and complement pathway component C1q-treated inflammatory reactive astrocytes, with significant overlap of differentially expressed genes. By contrasting ALS changes with models of protective reactive astrocytes, including middle cerebral artery occlusion and spinal cord injury, we uncover a cluster of genes changing in opposing directions, which may represent down-regulated homeostatic genes and up-regulated deleterious genes in ALS astrocytes. These observations indicate that ALS astrocytes augment inflammatory processes while concomitantly suppressing neuronal supporting mechanisms, thus resembling inflammatory reactive states and offering potential therapeutic targets.
A widely accepted tenet of evolutionary biology is that spontaneous mutations occur randomly with regard to their fitness effect. However, since the mutation rate varies along a genome and this ...variation can be subject to selection, organisms might evolve lower mutation rates at loci where mutations are most deleterious or increased rates where mutations are most needed. In fact, mechanisms of targeted hypermutation are known in organisms ranging from bacteria to humans. Here we review the main forces driving the evolution of local mutation rates and identify the main limiting factors. Both targeted hyper‐ and hypomutation can evolve, although the former is restricted to loci under very frequent positive selection and the latter is severely limited by genetic drift. Nevertheless, we show how an association of repair with transcription or chromatin‐associated proteins could overcome the drift limit and lead to non‐random hypomutation along the genome in most organisms.
Editor's suggested further reading in BioEssays Stress‐induced mutation via DNA breaks in Escherichia coli: A molecular mechanism with implications for evolution and medicine
Mutations are traditionally assumed to occur randomly with respect to fitness. However, mutation rates can vary along a genome and this variation can be evolutionarily optimized under certain conditions. Here we discuss the conditions and mechanisms by which selectively favourable mutational cold and hot spots can evolve along a genome.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do ...not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type-restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Abstract
Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal ...degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.
Dysregulation of proteostasis and RNA metabolism are increasingly recognised as critical drivers of neurodegenerative disease. Rafiee et al. focus on RNA polymerase II as the converging point of these two processes, and examine how transcriptional machinery and alternative splicing are affected by ALS-associated VCP mutations.
Understanding the molecular mechanisms and evolution of the gene regulatory system remains a major challenge in biology. Transcription start sites (TSSs) are especially interesting because they are ...central to initiating gene expression. Previous studies revealed widespread transcription initiation and fast turnover of TSSs in mammalian genomes. Yet, how new TSSs originate and how they evolve over time remain poorly understood. To address these questions, we analyzed ∼200,000 human TSSs by integrating evolutionary (inter- and intra-species) and functional genomic data, particularly focusing on evolutionarily young TSSs that emerged in the primate lineage. TSSs were grouped according to their evolutionary age using sequence alignment information as a proxy. Comparisons of young and old TSSs revealed that (1) new TSSs emerge through a combination of intrinsic factors, like the sequence properties of transposable elements and tandem repeats, and extrinsic factors such as their proximity to existing regulatory modules; (2) new TSSs undergo rapid evolution that reduces the inherent instability of repeat sequences associated with a high propensity of TSS emergence; and (3) once established, the transcriptional competence of surviving TSSs is gradually enhanced, with evolutionary changes subject to temporal (fewer regulatory changes in younger TSSs) and spatial constraints (fewer regulatory changes in more isolated TSSs). These findings advance our understanding of how regulatory innovations arise in the genome throughout evolution and highlight the genomic robustness and evolvability in these processes.
Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these--H-NS and ...Fis--bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.
Abstract
The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences ...of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3′ UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3′ UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions.
Gene Loops Enhance Transcriptional Directionality Tan-Wong, Sue Mei; Zaugg, Judith B.; Camblong, Jurgi ...
Science (American Association for the Advancement of Science),
11/2012, Volume:
338, Issue:
6107
Journal Article
Peer reviewed
Open access
Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from bidirectional promoters ...(nucleosome-depleted chromatin) that synthesize mRNA and ncRNA in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene-loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.
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