Alternative Pre-messenger RNA Research Articles

Alternative splicing links histone modifications to stem cell fate decision

BackgroundUnderstanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-messenger RNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear.ResultsWe analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended datasets from Roadmap/ENCODE projects, we exemplify this mechanism by a cell-cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1.ConclusionWe reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein-RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

AUF1 regulation of coding and noncoding RNA.

AUF1 is a family of four RNA-binding proteins (RBPs) generated by alternative pre-messenger RNA (pre-mRNA) splicing, with canonical roles in controlling the stability and/or translation of mRNA targets based on recognition of AU-rich sequences within mRNA 3' untranslated regions. However, recent studies identifying AUF1 target sites across the transcriptome have revealed that these canonical functions are but a subset of its roles in posttranscriptional regulation of gene expression. In this review, we describe recent developments in our understanding of the RNA-binding properties of AUF1 together with their biochemical implications and roles in directing mRNA decay and translation. This is then followed by a survey of newly discovered activities for AUF1 proteins in control of miRNA synthesis and function, including miRNA assembly into microRNA (miRNA)-loaded RNA-induced silencing complexes (miRISCs), miRISC targeting to mRNA substrates, interplay with an expanding network of other cellular RBPs, and reciprocal regulatory relationships between miRNA and AUF1 synthesis. Finally, we discuss recently reported relationships between AUF1 and long noncoding RNAs and regulatory roles on viral RNA substrates. Cumulatively, these findings have significantly expanded our appreciation of the scope and diversity of AUF1 functions in the cell, and are prompting an exciting array of new questions moving forward. WIREs RNA 2017, 8:e1393. doi: 10.1002/wrna.1393 For further resources related to this article, please visit the WIREs website.

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process.

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

Sudemycin E influences alternative splicing and changes chromatin modifications

Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death.

The Sam68 STAR RNA-Binding Protein Regulates mTOR Alternative Splicing during Adipogenesis

We report that mice ablated for the Sam68 RNA-binding protein exhibit a lean phenotype as a result of increased energy expenditure, decreased commitment to early adipocyte progenitors, and defects in adipogenic differentiation. The Sam68(-/-) mice were protected from obesity, insulin resistance, and glucose intolerance induced with a high-fat diet. To identify the alternative splice events regulated by Sam68, genome-wide exon usage profiling in white adipose tissue was performed. Adipocytes from Sam68(-/-) mice retained intron 5 within the mTOR transcript introducing a premature termination codon, leading to an unstable mRNA. Consequently, Sam68-depleted cells had reduced mTOR levels resulting in lower levels of insulin-stimulated S6 and Akt phosphorylation leading to defects in adipogenesis, and this defect was rescued by the exogenous expression of full-length mTOR. Sam68 bound intronic splice elements within mTOR intron 5 required for the usage of the 5' splice site. We propose that Sam68 regulates alternative splicing during adipogenesis.

High Resolution Imaging Via SHREC And SHRImP For Ultra-High DNA/RNA Resolution

Single-molecule high resolution co-localization (SHREC) (Churchman et al., PNAS, 2005) and Single-molecule high-resolution imaging with photobleaching (SHRImP) (Gordon et al., PNAS, 2004) methods have been developed to measure distances between two fluorophores that are closer than Rayleigh limit (≈250 nm for visible excitation). Combining the two techniques adds another dimension to the power of localization methodology and tens of distances could potentially be resolved by using several fluorophores of different colors each having multiple members.To apply this to DNA, we first stretched double-stranded DNA on a Polyacrylic acid and Polyallylamine coated surface, making the DNA relatively straight. To test SHRIMP, we made a DNA construct with a biotin followed by three Cy-3's at positions 475 bp, 172 bp, and 94 bp, corresponding to distances between Cy3 of 32nm, 58nm, and 90nm. We measured distances of 27 nm, 61nm, and 95 nm, in excellent agreement with the expected distances. To test simultaneously SHRIMP and SHREC, we placed Cy5 at position zero, and two Cy3's at position 94 bp and position 172 bp, and measured their positions using a dual-view imaging system. We determined the distances between Cy3-Cy5 pairs to be 37±5 nm (32 nm expected) and 91±5 nm (87 nm expected), and the distance between Cy3-Cy3 pair to be 56±3 nm (58 nm expected). The agreement is excellent. The next step in this project is to study alternative pre-messenger RNA splicing and to quantify individual splicing variants.

Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines

Alternative pre–messenger RNA splicing impacts development, physiology, and disease, but its regulation in humans is not well understood, partially due to the limited scale to which the expression of specific splicing events has been measured. We generated the first genome-scale expression compendium of human alternative splicing events using custom whole-transcript microarrays monitoring expression of 24,426 alternative splicing events in 48 diverse human samples. Over 11,700 genes and 9,500 splicing events were differentially expressed, providing a rich resource for studying splicing regulation. An unbiased, systematic screen of 21,760 4-mer to 7-mer words for cis-regulatory motifs identified 143 RNA 'words' enriched near regulated cassette exons, including six clusters of motifs represented by UCUCU, UGCAUG, UGCU, UGUGU, UUUU, and AGGG, which map to trans-acting regulators PTB, Fox, Muscleblind, CELF/CUG-BP, TIA-1, and hnRNP F/H, respectively. Each cluster showed a distinct pattern of genomic location and tissue specificity. For example, UCUCU occurs 110 to 35 nucleotides preceding cassette exons upregulated in brain and striated muscle but depleted in other tissues. UCUCU and UGCAUG appear to have similar function but independent action, occurring 5' and 3', respectively, of 33% of the cassette exons upregulated in skeletal muscle but co-occurring for only 2%.

Genome-wide detection of testis- and testicular cancer-specific alternative splicing

Alternative pre-messenger RNA (mRNA) splicing is a key molecular event that allows for protein diversity and plays important roles in development and disease. Alternative pre-mRNA splicing regulations during spermatogenesis and alternative pre-mRNA splicing etiology in testicular tumorigenesis are yet to be characterized. By genome-wide analysis, here we describe alternative splicing features that distinguish distinctive patterns of alternative pre-mRNA splicing among human testis, testicular cancer and mouse testis. Through computationally subtractive analysis, we detected 80 testis-specific transcript candidates in human testis, 175 in human testicular cancer and 262 in mouse testis, which were integrated into a database. Reverse transcription-polymerase chain reaction confirmed that most of these transcript candidates from mouse testis were testis specific. Around 40% of the transcripts were from unknown/hypothetical genes, which were useful for further functional analysis. These transcripts were not overlapped, indicating lack of evolutionary conservation. Further chromosome mapping showed distinct chromosomal preference of alternative pre-mRNA splicing events. Comparison analysis indicated that alternative pre-mRNA splicing in human testicular tumor shared some characters/trends with those in mouse testis. Moreover, human testicular tumor tended to use rare splice sites and there were also distinct sequences adjacent dominant splice sites between normal testis and testicular tumor. These special features of alternative pre-mRNA splicing in human testicular tumor suggested that testicular tumorigenesis was involved in multiple steps/levels of alternative splicing events. Using alternative splicing as a potential source for new clinical diagnostic, prognostic and therapeutic strategies for treatment of testicular tumors seems to have a bright prospect.

Alternative Splicing Matters: N-Type Calcium Channels in Nociceptors

How many different calcium channels does it take to make a nervous system? The answer: more than any of us predicted. In 1975 Hagiwara and colleagues published the first evidence that functionally different calcium channels are expressed in cells 1. By 1999, the calcium channel family could boast ten members, each member defined by a unique set of attributes to support their cellular functions and by unique amino acid sequences 2. Although nine of these genes are expressed in the nervous system, that number still seemed insufficient to support the wide spectrum of neuronal functions controlled by voltage-gated calcium channels. This discrepancy is probably explained by alternative pre-messenger RNA splicing which substantially expands the number of protein activities available from a limited number of genes. Like many other ion channel genes, each CaVa1 gene has the capacity to generate perhaps thousands of unique splice isoforms with unique functional properties 3. The high level of conservation among alternatively spliced exons in CaV2.2 genes of different species and in some cases closely related genes implies biological importance. A number of CaVa1 isoforms have been identified from neural tissue but until recently we lacked direct evidence linking a specific splice site in a calcium channel gene to a specific function in an identified neuron population.

The implications of alternative splicing in the ENCODE protein complement.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”

productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. [13][1]; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (

Splicing Regulation in Neurologic Disease

The importance of alternative splicing in the regulation of diverse biological processes is reflected in the growing list of human diseases associated with known or suspected splicing defects. It is becoming evident that alternative splicing plays a particularly important role in neurologic disease, which is perhaps not surprising given the important role splicing plays in generating complexity and function in the brain. This review considers the evidence that defects in regulation of splicing may underlie many types of human neurologic diseases.

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

Reprogramming Alternative Pre-messenger RNA Splicing through the Use of Protein-binding Antisense Oligonucleotides

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.

Single molecule profiling of alternative pre-mRNA splicing.

Alternative pre-messenger RNA splicing is an important mechanism for generating protein diversity and may explain in part how mammalian complexity arises from a surprisingly small complement of genes. Here, we describe "digital polony exon profiling,"a single molecule-based technology for studying complex alternative pre-messenger RNA splicing. This technology allows researchers to monitor the combinatorial diversity of exon inclusion in individual transcripts. A minisequencing strategy provides single nucleotide resolution, and the digital nature of the technology allows quantitation of individual splicing variants. Digital polony exon profiling can be used to investigate the physiological and pathological roles of alternately spliced messenger RNAs, as well as the mechanisms by which these messenger RNAs are produced.

Mechanisms of Alternative Pre-Messenger RNA Splicing

Alternative pre-mRNA splicing is a central mode of genetic regulation in higher eukaryotes. Variability in splicing patterns is a major source of protein diversity from the genome. In this review, I describe what is currently known of the molecular mechanisms that control changes in splice site choice. I start with the best-characterized systems from the Drosophila sex determination pathway, and then describe the regulators of other systems about whose mechanisms there is some data. How these regulators are combined into complex systems of tissue-specific splicing is discussed. In conclusion, very recent studies are presented that point to new directions for understanding alternative splicing and its mechanisms.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murine survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin(140), similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin(121) that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin(40), lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin(140) and survivin(121) inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin(140) mRNA, whereas survivin(121)-specific transcripts were detected in all tissues, while those representing survivin(40) were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis. (Blood. 2000;95:1435-1442)

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins ( su( w a ) and tra) that may regulate alternative pre-messenger RNA splicing.