pre-mRNA Molecules Research Articles

SmFRET-assisted RNA structure prediction.

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful biophysical technique that utilizes the distance-dependent energy transfer between donor and acceptor dyes linked to individual molecules, providing insights into molecular conformational changes and interactions at the single-molecule level. Prior investigations leveraged smFRET to study the conformational dynamics of single truncated Ubc4 pre-mRNA molecules during splicing, yet these efforts did not prioritize structural modeling. In this study, we develop an smFRET-assisted RNA prediction method to predict the 2D and 3D structures of this pre-mRNA. To achieve this, we initiate the process by generating RNA structural ensembles through coarse-grained molecular dynamics (MD) simulations. Subsequently, inter-dye distances are calculated for these RNA structural ensembles by performing all-atom MD simulations of the dye groups. The ultimate determination of the 2D and 3D structures for the pre-mRNA is achieved by comparing the calculated inter-dye distances with experimental counterparts. Notably, our computational results demonstrate a significant alignment with experimental findings, which involve a conformational change at the 2D level.

Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation

RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.

Fibronectin and Its Applications in Dentistry and Periodontics: A Cell Behaviour Conditioner.

The formation of biomaterials is a physical phenomenon that is primarily influenced by the material's chemical and physical characteristics, as well as by the availability of proteins and their mutual interactions. A common extracellular matrix (ECM) glycoprotein called fibronectin (FN) is a biomaterial that is essential for tissue repair. Cellular FN (cFN), also known as the "large external transformation sensitive (LETS) protein" or "galactoprotein,"was found during the quest for tumour markers twenty-five years ago and was later identified as the surface fibroblast antigen. Twenty different isoforms of the FN protein can be created by alternative splicing of a single pre-messenger ribonucleic acid (pre-mRNA) molecule. FN is an outstanding illustration of an ECM protein that intricately influences cell activity. FN is necessary for cell behaviours like cell adhesion, cell migration, and differentiation of cells as well as highly coordinated tissue processes like morphogenesis and wound repair. Plasma FN is absorbed by tissues and deposited in extracellular matrix fibrils along with locally generated cellular FN. cFN is produced by a wide range of cell types, including fibroblasts, endothelial cells, chondrocytes, synovial cells, and myocytes. FN and other cell adhesion proteins can promote cell attachment to tooth surfaces. Periodontal ligament (PDL) cell-ECM interactions, and consequently the regeneration of periodontal tissues, depends on FN. Specific FN segments serve as indicators of periodontal disease status and provide evidence for their potential involvement in the pathophysiology of the condition. FN is an all-purpose biomaterialthat may be utilised for clinical applications ranging from tissue engineering to disease biology. Therefore, it would be desirableto develop materials that specifically bind to FN.

Non-Canonical Splicing and Its Implications in Brain Physiology and Cancer.

The advance of experimental and computational techniques has allowed us to highlight the existence of numerous different mechanisms of RNA maturation, which have been so far unknown. Besides canonical splicing, consisting of the removal of introns from pre-mRNA molecules, non-canonical splicing events may occur to further increase the regulatory and coding potential of the human genome. Among these, splicing of microexons, recursive splicing and biogenesis of circular and chimeric RNAs through back-splicing and trans-splicing processes, respectively, all contribute to expanding the repertoire of RNA transcripts with newly acquired regulatory functions. Interestingly, these non-canonical splicing events seem to occur more frequently in the central nervous system, affecting neuronal development and differentiation programs with important implications on brain physiology. Coherently, dysregulation of non-canonical RNA processing events is associated with brain disorders, including brain tumours. Herein, we summarize the current knowledge on molecular and regulatory mechanisms underlying canonical and non-canonical splicing events with particular emphasis on cis-acting elements and trans-acting factors that all together orchestrate splicing catalysis reactions and decisions. Lastly, we review the impact of non-canonical splicing on brain physiology and pathology and how unconventional splicing mechanisms may be targeted or exploited for novel therapeutic strategies in cancer.

The Fine Art of Writing a Message: RNA Metabolism in the Shaping and Remodeling of the Nervous System.

Neuronal morphogenesis, integration into circuits, and remodeling of synaptic connections occur in temporally and spatially defined steps. Accordingly, the expression of proteins and specific protein isoforms that contribute to these processes must be controlled quantitatively in time and space. A wide variety of post-transcriptional regulatory mechanisms, which act on pre-mRNA and mRNA molecules contribute to this control. They are thereby critically involved in physiological and pathophysiological nervous system development, function, and maintenance. Here, we review recent findings on how mRNA metabolism contributes to neuronal development, from neural stem cell maintenance to synapse specification, with a particular focus on axon growth, guidance, branching, and synapse formation. We emphasize the role of RNA-binding proteins, and highlight their emerging roles in the poorly understood molecular processes of RNA editing, alternative polyadenylation, and temporal control of splicing, while also discussing alternative splicing, RNA localization, and local translation. We illustrate with the example of the evolutionary conserved Musashi protein family how individual RNA-binding proteins are, on the one hand, acting in different processes of RNA metabolism, and, on the other hand, impacting multiple steps in neuronal development and circuit formation. Finally, we provide links to diseases that have been associated with the malfunction of RNA-binding proteins and disrupted post-transcriptional regulation.

A broad analysis of splicing regulation in yeast using a large library of synthetic introns.

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression.

Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5′ and 3′ ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5′ and 3′ ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.

Stochastic Gene Expression Revisited.

We investigate the model of gene expression in the form of Iterated Function System (IFS), where the probability of choice of any iterated map depends on the state of the phase space. Random jump times of the process mark activation periods of the gene when pre-mRNA molecules are produced before mRNA and protein processing phases occur. The main idea is inspired by the continuous-time piecewise deterministic Markov process describing stochastic gene expression. We show that for our system there exists a unique invariant limit measure. We provide full probabilistic description of the process with a comparison of our results to those obtained for the model with continuous time.

Computational analysis of spliced leader trans-splicing in the regenerative flatworm Macrostomum lignano reveals its prevalence in conserved and stem cell related genes.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and significantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

Regulation of the parental gene GRM4 by circGrm4 RNA transcript and glutamate-mediated neurovascular toxicity in eyes.

Epigenetic memory plays crucial roles in gene regulation. It not only modulates the expression of specific genes but also has ripple effects on transcription as well as translation of other genes. Very often an alteration in expression occurs either via methylation or demethylation. In this context, "1-carbon metabolism" assumes a special significance since its dysregulation by higher levels of homocysteine; Hcy (known as hyperhomocysteinemia; HHcy), a byproduct of "1-Carbon Metabolism" during methionine biosynthesis leads to serious implications in cardiovascular, renal, cerebrovascular systems, and a host of other conditions. Currently, the circular RNAs (circRNAs) generated via non-canonical back-splicing events from the pre-mRNA molecules are at the center stage for their essential roles in diseases via their epigenetic manifestations. We recently identified a circular RNA transcript (circGRM4) that is significantly upregulated in the eye of cystathionine β-synthase-deficient mice. We also discovered a concurrent over-expression of the mGLUR4 receptor in the eyes of these mice. In brief, circGRM4 is selectively transcribed from its parental mGLUR4 receptor gene (GRM4) functions as a "molecular-sponge" for the miRNAs and results into excessive turnover of the mGLUR4 receptor in the eye in response to extremely high circulating glutamate concentration. We opine that this epigenetic manifestation potentially predisposes HHcy people to retinovascular malfunctioning.

The Role of RNA Splicing Factors in Cancer: Regulation of Viral and Human Gene Expression in Human Papillomavirus-Related Cervical Cancer.

The spliceosomal complex components, together with the heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins, regulate the process of constitutive and alternative splicing, the latter leading to the production of mRNA isoforms coding multiple proteins from a single pre-mRNA molecule. The expression of splicing factors is frequently deregulated in different cancer types causing the generation of oncogenic proteins involved in cancer hallmarks. Cervical cancer is caused by persistent infection with oncogenic human papillomaviruses (HPVs) and constitutive expression of viral oncogenes. The aberrant activity of hnRNPs and SR proteins in cervical neoplasia has been shown to trigger the production of oncoproteins through the processing of pre-mRNA transcripts either derived from human genes or HPV genomes. Indeed, hnRNP and SR splicing factors have been shown to regulate the production of viral oncoprotein isoforms necessary for the completion of viral life cycle and for cell transformation. Target-therapy strategies against hnRNPs and SR proteins, causing simultaneous reduction of oncogenic factors and inhibition of HPV replication, are under development. In this review, we describe the current knowledge of the functional link between RNA splicing factors and deregulated cellular as well as viral RNA maturation in cervical cancer and the opportunity of new therapeutic strategies.

Nucleotide Modifications Decrease Innate Immune Response Induced by Synthetic Analogs of snRNAs and snoRNAs.

Short nuclear regulatory RNAs play a key role in the main stages of maturation of the precursors of the major RNA species. Small nuclear RNAs (snRNAs) form the core of the spliceosome and are responsible for the splicing of pre-mRNA molecules. Small nucleolar RNAs (snoRNAs) direct post-transcriptional modification of pre-rRNAs. A promising strategy for the development of non-coding RNA (ncRNAs) mimicking molecules is the introduction of modified nucleotides, which are normally present in natural ncRNAs, into the structure of synthetic RNAs. We have created a set of snoRNAs and snRNA analogs and studied the effect of base modifications, specifically, pseudouridine (Ψ) and 5-methylcytidine (m5C), on the immune-stimulating and cytotoxic properties of these RNAs. Here, we performed a whole-transcriptome study of the influence of synthetic snoRNA analogs with various modifications on gene expression in human cells. Moreover, we confirmed the role of PKR in the recognition of snoRNA and snRNA analogs using the short hairpin RNA (shRNA) technique. We believe that the data obtained will contribute to the understanding of the role of nucleotide modification in ncRNA functions, and can be useful for creating the agents for gene regulation based on the structure of natural snoRNAs and snRNAs.

Alternative Splicing as a Regulator of Early Plant Development.

Most plant genes are interrupted by introns and the corresponding transcripts need to undergo pre-mRNA splicing to remove these intervening sequences. Alternative splicing (AS) is an important posttranscriptional process that creates multiple mRNA variants from a single pre-mRNA molecule, thereby enhancing the coding and regulatory potential of genomes. In plants, this mechanism has been implicated in the response to environmental cues, including abiotic and biotic stresses, in the regulation of key developmental processes such as flowering, and in circadian timekeeping. The early plant development steps – from embryo formation and seed germination to skoto- and photomorphogenesis – are critical to both execute the correct body plan and initiate a new reproductive cycle. We review here the available evidence for the involvement of AS and various splicing factors in the initial stages of plant development, while highlighting recent findings as well as potential future challenges.

SL-quant: a fast and flexible pipeline to quantify spliced leader trans-splicing events from RNA-seq data.

BackgroundThe spliceosomal transfer of a short spliced leader (SL) RNA to an independent pre-mRNA molecule is called SL trans-splicing and is widespread in the nematode Caenorhabditis elegans. While RNA-sequencing (RNA-seq) data contain information on such events, properly documented methods to extract them are lacking.FindingsTo address this, we developed SL-quant, a fast and flexible pipeline that adapts to paired-end and single-end RNA-seq data and accurately quantifies SL trans-splicing events. It is designed to work downstream of read mapping and uses the reads left unmapped as primary input. Briefly, the SL sequences are identified with high specificity and are trimmed from the input reads, which are then remapped on the reference genome and quantified at the nucleotide position level (SL trans-splice sites) or at the gene level.Conclusions SL-quant completes within 10 minutes on a basic desktop computer for typical C. elegans RNA-seq datasets and can be applied to other species as well. Validating the method, the SL trans-splice sites identified display the expected consensus sequence, and the results of the gene-level quantification are predictive of the gene position within operons. We also compared SL-quant to a recently published SL-containing read identification strategy that was found to be more sensitive but less specific than SL-quant. Both methods are implemented as a bash script available under the MIT license [1]. Full instructions for its installation, usage, and adaptation to other organisms are provided.

Synergistic assembly of human pre-spliceosomes across introns and exons.

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

The emerging role of minor intron splicing in neurological disorders.

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5' and 3' ends and are recognized by a separate pre-mRNA processing machinery denoted as the minor spliceosome. This spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on minor spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem–loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X.

Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5′ splice site (5′SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5′SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5′SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5′SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.

The effects of structure on pre-mRNA processing and stability.

Pre-mRNA molecules can form a variety of structures, and both secondary and tertiary structures have important effects on processing, function and stability of these molecules. The prediction of RNA secondary structure is a challenging problem and various algorithms that use minimum free energy, maximum expected accuracy and comparative evolutionary based methods have been developed to predict secondary structures. However, these tools are not perfect, and this remains an active area of research. The secondary structure of pre-mRNA molecules can have an enhancing or inhibitory effect on pre-mRNA splicing. An example of enhancing structure can be found in a novel class of introns in zebrafish. About 10% of zebrafish genes contain a structured intron that forms a bridging hairpin that enforces correct splice site pairing. Negative examples of splicing include local structures around splice sites that decrease splicing efficiency and potentially cause mis-splicing leading to disease. Splicing mutations are a frequent cause of hereditary disease. The transcripts of disease genes are significantly more structured around the splice sites, and point mutations that increase the local structure often cause splicing disruptions. Post-splicing, RNA secondary structure can also affect the stability of the spliced intron and regulatory RNA interference pathway intermediates, such as pre-microRNAs. Additionally, RNA secondary structure has important roles in the innate immune defense against viruses. Finally, tertiary structure can also play a large role in pre-mRNA splicing. One example is the G-quadruplex structure, which, similar to secondary structure, can either enhance or inhibit splicing through mechanisms such as creating or obscuring RNA binding protein sites.

The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules.

Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.