G-Quadruplexes in RNA Biology: Recent Advances and Future Directions.

RNA G-quadruplexes (RG4s) are four-stranded structures known to control gene expression mechanisms, from transcription to protein synthesis, and DNA-related processes. Their potential impact on RNA biology allows these structures to shape cellular processes relevant to disease development, making their targeting for therapeutic purposes an attractive option. We review here the current knowledge on RG4s, focusing on the latest breakthroughs supporting the notion of transient structures that fluctuate dynamically in cellulo, their interplay with RNA modifications, their role in cell compartmentalization, and their deregulation impacting the host immune response. We emphasize RG4-binding proteins as determinants of their transient conformation and effectors of their biological functions

A physical and functional link between splicing factors promotes pre-mRNA 3′ end processing

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3′ end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3′ end formation. We identify PTB as the protein factor whose binding to the human β-globin (HBB) 3′ UTR is abrogated by a 3′ end processing-inactivating mutation. We show that PTB promotes both in vitro 3′ end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements

Regulatory mechanisms for 3′-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

The glial fibrillary acidic protein, GFAP, forms the intermediate cytoskeleton in cells of the glial lineage. Besides the common GFAPα transcript, the GFAPε and GFAPκ transcripts are generated by alternative mRNA 3′-end processing. Here we use a GFAP minigene to characterize molecular mechanisms participating in alternative GFAP expression. Usage of a polyadenylation signal within the alternatively spliced exon 7a is essential to generate the GFAPκ and GFAPκ transcripts. The GFAPκ mRNA is distinct from GFAPε mRNA given that it also includes intron 7a. Polyadenylation at the exon 7a site is stimulated by the upstream splice site. Moreover, exon 7a splice enhancer motifs supported both exon 7a splicing and polyadenylation. SR proteins increased the usage of the exon 7a polyadenylation signal but not the exon 7a splicing, whereas the polypyrimidine tract binding (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model with the selection of the exon 7a polyadenylation site being the essential and primary event for regulating GFAP alternative processing

AltAnalyze and DomainGraph: analyzing and visualizing exon expression data

Alternative splicing is an important mechanism for increasing protein diversity. However, its functional effects are largely unknown. Here, we present our new software workflow composed of the open-source application AltAnalyze and the Cytoscape plugin DomainGraph. Both programs provide an intuitive and comprehensive end-to-end solution for the analysis and visualization of alternative splicing data from Affymetrix Exon and Gene Arrays at the level of proteins, domains, microRNA binding sites, molecular interactions and pathways. Our software tools include easy-to-use graphical user interfaces, rigorous statistical methods (FIRMA, MiDAS and DABG filtering) and do not require prior knowledge of exon array analysis or programming. They provide new methods for automatic interpretation and visualization of the effects of alternative exon inclusion on protein domain composition and microRNA binding sites. These data can be visualized together with affected pathways and gene or protein interaction networks, allowing a straightforward identification of potential biological effects due to alternative splicing at different levels of granularity. Our programs are available at http://www.altanalyze.org and http://www.domaingraph.de. These websites also include extensive documentation, tutorials and sample data

Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation

Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3′ end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3′ end processing regulation by proposing several overlapping models of regulation

The type II poly(A)-binding protein PABP-2 genetically interacts with the let-7 miRNA and elicits heterochronic phenotypes in Caenorhabditis elegans

The type II poly(A)-binding protein PABP2/PABPN1 functions in general mRNA metabolism by promoting poly(A) tail formation in mammals and flies. It also participates in poly(A) tail shortening of specific mRNAs in flies, and snoRNA biogenesis in yeast. We have identified Caenorhabditis elegans pabp-2 as a genetic interaction partner of the let-7 miRNA, a widely conserved regulator of animal stem cell fates. Depletion of PABP-2 by RNAi suppresses loss of let-7 activity, and, in let-7 wild-type animals, leads to precocious differentiation of seam cells. This is not due to an effect on let-7 biogenesis and activity, which remain unaltered. Rather, PABP-2 levels are developmentally regulated in a let-7-dependent manner. Moreover, using RNAi PABP-2 can be depleted by >80% without significantly impairing larval viability, mRNA levels or global translation. Thus, it unexpectedly appears that the bulk of PABP-2 is dispensable for general mRNA metabolism in the larva and may instead have more restricted, developmental functions. This observation may be relevant to our understanding of why the phenotypes associated with human PABP2 mutation in oculopharyngeal muscular dystrophy (OPMD) seem to selectively affect only muscle cells

Crystal structure of the 25 kDa subunit of human cleavage factor Im

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts

Background: Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response

Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3′End Processing in Magnaporthe oryzae

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3′UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3′end processing mechanisms exclusive to the fungal kingdom