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1.  Mutations in the Caenorhabditis elegans U2AF Large Subunit UAF-1 Alter the Choice of a 3′ Splice Site In Vivo 
PLoS Genetics  2009;5(11):e1000708.
The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the in vivo regulation of splicing. From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3′ splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3′ splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3′ splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3′ splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3′ splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3′ splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans.
Author Summary
Eukaryotic genes contain intervening intronic sequences that must be removed from pre-mRNA transcripts by RNA splicing to generate functional messenger RNAs. While studying genes that encode and control a presumptive muscle potassium channel complex in the nematode Caenorhabditis elegans, we found that mutations in two splicing factors, the U2AF large subunit and SF1/BBP suppress the rubberband Unc phenotype caused by a rare missense mutation in the gene unc-93. Mutations affecting the U2AF large subunit caused the recognition of a cryptic 3′ splice site generated by the unc-93 mutation, providing in vivo evidence that the U2AF large subunit can affect splice-site selection. By contrast, an SF1/BBP mutation that suppressed the rubberband Unc phenotype did not cause splicing using this cryptic 3′ splice site. Our genetic studies identified a region of the U2AF large subunit important for its effect on 3′ splice-site choice. Our mutagenesis analysis of in vivo transgene splicing identified a positional effect on weak 3′ splice site selection and nucleotides of the endogenous 3′ splice site important for recognition. The system we have defined should facilitate future in vivo analyses of pre–mRNA splicing.
doi:10.1371/journal.pgen.1000708
PMCID: PMC2762039  PMID: 19893607
2.  The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing 
PLoS Biology  2011;9(1):e1000573.
Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.
RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.
Author Summary
The pre-mRNA emerging from RNA polymerase II during eukaryotic transcription undergoes a series of processing events. These include 5′-capping, intron excision and exon ligation during splicing, 3′-end processing, and polyadenylation. Processing events occur co-transcriptionally, meaning that a variety of enzymes assemble on the pre-mRNA while the polymerase is still engaged in transcription. The concept of co-transcriptional mRNA processing raises questions about the possible coupling between the transcribing polymerase and the processing machineries. Here we examine how the co-transcriptional assembly of the splicing machinery (the spliceosome) might affect the elongation kinetics of the RNA polymerase. Using live-cell microscopy, we followed the kinetics of transcription of genes containing increasing numbers of introns and measured the recruitment of transcription and splicing factors. Surprisingly, a sub-set of splicing factors was recruited to an intronless gene, implying that there is a polymerase-coupled scanning mechanism for intronic sequences. There was no difference in polymerase elongation rates on genes with or without introns, suggesting that the spliceosome does not modulate elongation kinetics. Experiments including inhibition of splicing or transcription, together with stochastic computational simulation, demonstrated that pre-mRNAs can be retained on the gene when polymerase termination precedes completion of splicing. Altogether we show that polymerase elongation kinetics are not affected by splicing events on the emerging pre-mRNA, that increased splicing leads to more splicing factors being recruited to the mRNA, and that post-transcriptional splicing can proceed at the site of transcription in the absence of the polymerase.
doi:10.1371/journal.pbio.1000573
PMCID: PMC3019111  PMID: 21264352
3.  Oriented Scanning Is the Leading Mechanism Underlying 5′ Splice Site Selection in Mammals 
PLoS Genetics  2006;2(9):e138.
Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection.
Synopsis
Typically, mammalian genes contain coding sequences (exons) separated by non-coding sequences (introns). Introns are removed during pre-mRNA splicing. The accurate recognition of introns during splicing is essential, as any abnormality in that process will generate abnormal mRNAs that can cause diseases. Understanding the mechanisms of accurate splice site selection is of prime interest to life scientists. Exon–intron borders (splice sites) are defined by short sequences that are poorly conserved. The strength of any splice sequence can be assessed by its degree of homology with a splice site consensus sequence. Within exons and introns, several sequences can match with this consensus as well as or better than the splice sites. Using a system in which a splice site sequence is repeated several times in the intron, the authors showed that linear 5′−3′ search is a leading mechanism underlying splice site selection. This scanning mechanism is cell type–independent, and only the most upstream splice site of all the series is selected, even if splice sites with a better match to the consensus are in the vicinity. These findings reconciliate contradictory observations and establish a hierarchy among the determinants involved in splice site selection.
doi:10.1371/journal.pgen.0020138
PMCID: PMC1557585  PMID: 16948532
4.  A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression 
PLoS Genetics  2013;9(10):e1003856.
The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.
Author Summary
The accurate removal of intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) represents an essential step in the expression of most eukaryotic protein-coding genes. Alternative splicing can create from a single primary transcript various mature mRNAs with diverse, sometimes even antagonistic, biological functions. Many human diseases are based on alternative-splicing defects, and most interestingly, certain defects are caused by mutations in general splicing factors that participate in each splicing event. To address the question of how a general splicing factor can regulate alternative splicing events, here we investigated the regulatory role of the U1C protein, a specific component of the U1 small nuclear ribonucleoprotein (snRNP) and important in initial 5′ splice site recognition. Our RNA-Seq analysis demonstrated that U1C affects more than 300 cases of alternative splicing in the human system. One U1C target, U1-70K, appeared to be particularly interesting, because both protein products are components of the U1 snRNP and functionally depend on each other. Analyzing the mechanistic basis of this intra-U1 snRNP cross-regulation, we discovered a U1C-dependent alternative splicing switch in the U1-70K pre-mRNA that regulates U1-70K expression. In sum, this feedback loop controls and links U1C and U1-70K homeostasis to guarantee correct U1 snRNP assembly and function.
doi:10.1371/journal.pgen.1003856
PMCID: PMC3798272  PMID: 24146627
5.  Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing 
Molecular and Cellular Biology  2001;21(22):7673-7681.
The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF65) binds to the polypyrimidine (Py) tract preceding the 3′ splice site, while the 35-kDa subunit (U2AF35) contacts the conserved AG dinucleotide at the 3′ end of the intron. It has been shown that the interaction between U2AF35 and the 3′ splice site AG can stabilize U2AF65 binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF35 has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF65 binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF65. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3′ splice site AG, ESE) responsible for the U2AF35 dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF35 dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF35 function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF35 activity and because a truncation mutant of U2AF35 consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF35 can be explained in terms of enhanced U2AF65 binding, other activities of U2AF35 do not correlate with increased cross-linking of U2AF65 to the Py tract. Collectively, the results argue that interaction of U2AF35 with a consensus 3′ splice site triggers events in spliceosome assembly in addition to stabilizing U2AF65 binding, thus revealing a dual function for U2AF35 in pre-mRNA splicing.
doi:10.1128/MCB.21.22.7673-7681.2001
PMCID: PMC99938  PMID: 11604503
6.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements 
PLoS Genetics  2007;3(5):e85.
Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.
Author Summary
During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.
doi:10.1371/journal.pgen.0030085
PMCID: PMC1877881  PMID: 17530930
7.  Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing* 
The Journal of biological chemistry  2003;278(25):22740-22747.
The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5′-splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3′-splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3′-splice site choice. However, little is known about enhancer-dependent regulation of alternative 5′-splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5′-splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5′-splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5′- and 3′-splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5′- and 3′-splice sites.
doi:10.1074/jbc.M301036200
PMCID: PMC2386364  PMID: 12646561
8.  The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing 
PLoS Genetics  2012;8(7):e1002827.
RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing.
Author Summary
RNA splicing removes intervening intronic sequences from pre–mRNA transcripts and joins adjacent exonic sequences to generate functional messenger RNAs. The in vivo functions of numerous factors that regulate splicing remain to be understood. From a genetic screen for suppressors of the rubberband Unc phenotype caused by the Caenorhabditis elegans unc-93(e1500) mutation, we isolated a mutation that affects a highly conserved essential gene, mfap-1. MFAP-1 is a nuclear protein that is broadly expressed. MFAP-1 can affect the alternative splicing of tos-1, an endogenous reporter gene for splicing, and is required for the altered splicing at a cryptic 3′ splice site of tos-1. mfap-1 enhances the effects of the gene uaf-1 (splicing factor U2AF large subunit) in suppressing the rubberband Unc phenotype of unc-93(e1500) animals. Our studies provide in vivo evidence that MFAP-1 functions as a splicing factor.
doi:10.1371/journal.pgen.1002827
PMCID: PMC3400559  PMID: 22829783
9.  Exon definition may facilitate splice site selection in RNAs with multiple exons. 
Molecular and Cellular Biology  1990;10(1):84-94.
Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.
Images
PMCID: PMC360715  PMID: 2136768
10.  Cell-to-cell variability of alternative RNA splicing 
The role of mRNA processing in gene expression variability is poorly characterized. This study investigates the extent of cell-to-cell variability of alternative RNA splicing in mammalian cells using single-molecule imaging of CAPRIN1 and MKNK2 splice isoforms.
We applied a single-molecule imaging approach to visualize the alternatively spliced isoforms of two genes, CAPRIN1 and MKNK2, in human cells.We found that cell-to-cell variability in isoform ratios is close to the minimum possible in the absence of feedback in clonal Rpe1 cells, a diploid non-transformed cell line. In contrast, clonal HeLa cells displayed much larger isoform ratio variability between cells.Experimental and theoretical analysis suggests that variability in the regulatory splicing machinery contributes to this difference between cell lines.
Biological gene expression is a complex process which includes transcription, mRNA processing, and translation. As gene expression is a fundamental aspect of biological behavior, a central question within the fields of molecular and cellular biology is how effectively cells control the abundance of their gene expression products, mRNA and protein.
Previous experimental and theoretical studies have shown that there can be substantial cell-to-cell variation in gene expression, even between genetically identical cells grown in uniform conditions. This variation was shown to be important in a variety of biological contexts such as development, virology, immune system function, and cancer treatment. One major source of variability was shown to be transcriptional bursting, or the process in which genes are expressed sporadically separated by long durations of inexpression. Additionally, since the biochemical reactions that govern gene expression are often mediated by molecular species that are present in low numbers, variability can arise from stochastic effects owing to the random chance that an individual biochemical reaction will occur.
The role of mRNA processing in gene expression variability has not been examined thoroughly, particularly with respect to alternative splicing. Alternative RNA splicing is a form of mRNA processing which leads to the synthesis of multiple different mRNAs from a single gene. In this process, the nascent mRNA (pre-mRNA) of a gene contains sequences known as introns that can be excised in different combinations to generate multiple gene products, known as isoforms. As alternative splicing occurs in the vast majority of human genes, it presents a potentially major source of cell-to-cell variability in gene expression.
In this study, we sought to characterize the extent of cell-to-cell variability that arises from alternative RNA splicing. To do so, we utilized a single-molecule imaging approach based on fluorescent in situ hybridization to study the cell-to-cell variability in isoform ratios of two genes, CAPRIN1 and MKNK2, which each contain two splice isoforms (Figure 2 from the manuscript). Using a clonally derived, diploid, non-transformed cell line (Rpe1 cells—retinal pigment epithelial cells), we found that variability is remarkably close to the minimum possible given the probabilistic chance of individual splicing events. In contrast, we found that isoform ratio variability was substantially larger in clonally derived HeLa cells, a cancerous cell line with an unstable karyotype. To explain the differences between the two cell lines, we further examined the potential origins of isoform ratio variability. We first studied several known sources of mRNA variability, such as transcriptional bursting, but found that they did not contribute significantly to the difference between cell lines. However, when we examined the role of splicing factors in controlling cell-to-cell variability, we found that lesser control over the regulation of alternative splicing is likely to be the primary source of this difference.
Cell-to-cell variability in gene expression owing to alternative splicing is an inevitable feature of biology. Since spliced isoforms can have different and even opposing cellular functions, it would be interesting to see if such variability can have phenotypic consequences in various biological settings. We anticipate that future work will shed light on the extent of cell-to-cell variability of alternative splicing for additional genes, and may identify splicing events where heterogeneity has an important functional role.
Heterogeneity in the expression levels of mammalian genes is large even in clonal populations and has phenotypic consequences. Alternative splicing is a fundamental aspect of gene expression, yet its contribution to heterogeneity is unknown. Here, we use single-molecule imaging to characterize the cell-to-cell variability in mRNA isoform ratios for two endogenous genes, CAPRIN1 and MKNK2. We show that isoform variability in non-transformed, diploid cells is remarkably close to the minimum possible given the stochastic nature of individual splicing events, while variability in HeLa cells is considerably higher. Analysis of the potential sources of isoform ratio heterogeneity indicates that a difference in the control over splicing factor activity is one origin of this increase. Our imaging approach also visualizes non-alternatively spliced mRNA and active transcription sites, and yields spatial information regarding the relationship between splicing and transcription. Together, our work demonstrates that mammalian cells minimize fluctuations in mRNA isoform ratios by tightly regulating the splicing machinery.
doi:10.1038/msb.2011.32
PMCID: PMC3159976  PMID: 21734645
alternative splicing; cell-to-cell variability; co-transcriptional splicing; gene expression
11.  Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo 
Journal of Virology  2000;74(13):5902-5910.
Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.
PMCID: PMC112086  PMID: 10846071
12.  Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6. 
Nucleic Acids Research  1994;22(12):2318-2325.
We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.
Images
PMCID: PMC523690  PMID: 8036160
13.  Identification of motifs that function in the splicing of non-canonical introns 
Genome Biology  2008;9(6):R97.
The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif.
Background
While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.
Results
Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.
Conclusion
The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.
doi:10.1186/gb-2008-9-6-r97
PMCID: PMC2481429  PMID: 18549497
14.  Transcript Specificity in Yeast Pre-mRNA Splicing Revealed by Mutations in Core Spliceosomal Components 
PLoS Biology  2007;5(4):e90.
Appropriate expression of most eukaryotic genes requires the removal of introns from their pre–messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.
Author Summary
The spliceosome is a large RNA-protein machine responsible for removing the noncoding (intron) sequences that interrupt eukaryotic genes. Nearly everything known about the behavior of this machine has been based on the analysis of only a handful of genes, despite the fact that individual introns vary greatly in both size and sequence. Here we have utilized a microarray-based platform that allows us to simultaneously examine the behavior of all intron-containing genes in the budding yeast S. cerevisiae. By systematically examining the effects of individual mutants in the spliceosome on the splicing of all substrates, we have uncovered a surprisingly complex relationship between the spliceosome and its full complement of substrates. Contrary to the idea that the spliceosome engages in “generic” interactions with all intron-containing substrates in the cell, our results show that the identity of the transcript can differentially affect splicing efficiency when the machine is subtly perturbed. We propose that the wild-type spliceosome can also distinguish among its many substrates as external conditions warrant to function as a specific regulator of gene expression.
Many eukaryotic gene transcripts are spliced; here the authors show that components of the splicing complex can distinguish between different introns in highly homologous transcripts.
doi:10.1371/journal.pbio.0050090
PMCID: PMC1831718  PMID: 17388687
15.  Pre-mRNA Secondary Structures Influence Exon Recognition 
PLoS Genetics  2007;3(11):e204.
The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.
Author Summary
Almost all human protein-coding genes contain several exons and introns. Prior to translation, introns have to be removed and exons have to be joined, which happens in a processing step called splicing that generates the mature mRNA. For most genes, certain exons can be either included or excluded from the mature mRNA. It is currently not fully understood which signals are needed to accurately recognize the boundaries of exons in the intron-containing primary transcript. As in transcriptional regulation, enhancer and silencer sequence motifs are crucial for the correct recognition of exons. Splicing regulatory proteins identify these motifs in a sequence-specific manner. In general, these proteins bind to single-stranded RNA. Here, we analyzed local secondary structures of primary transcripts and found that known splicing motifs are preferentially located in a single-stranded context. Experimental tests demonstrated that motifs in single-stranded contexts have a stronger effect on splice site selection than those located in double-stranded regions. These results help to understand the action of human mutations that change the splicing pattern and indicate that local pre-mRNA secondary structures influence exon recognition.
doi:10.1371/journal.pgen.0030204
PMCID: PMC2077896  PMID: 18020710
16.  Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse 
PLoS ONE  2007;2(5):e427.
Background
Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5′ and 3′ splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described.
Methodology/Principal Finding
Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3′ splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24.
Conclusions/Significance
Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites.
doi:10.1371/journal.pone.0000427
PMCID: PMC1855434  PMID: 17487273
17.  Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing 
Human Molecular Genetics  2009;18(17):3266-3273.
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5′ splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic +14 mutation produce exclusively E10+ RNA. Generation of E10− RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the Δ280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.
doi:10.1093/hmg/ddp264
PMCID: PMC2722988  PMID: 19498037
18.  Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing▿  
Molecular and Cellular Biology  2010;30(8):1878-1886.
Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.
doi:10.1128/MCB.01071-09
PMCID: PMC2849477  PMID: 20123971
19.  Spliceosome Assembly Pathways for Different Types of Alternative Splicing Converge during Commitment to Splice Site Pairing in the A Complex▿ †  
Molecular and Cellular Biology  2008;29(4):1072-1082.
Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5′ splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATPγS, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point.
doi:10.1128/MCB.01071-08
PMCID: PMC2643811  PMID: 19064642
20.  Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation. 
Nucleic Acids Research  1994;22(3):332-337.
The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.
Images
PMCID: PMC523585  PMID: 8127670
21.  The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity 
Nucleic Acids Research  2002;30(21):4607-4617.
A novel class of RNA-binding proteins, Puf, regulates translation and RNA stability by binding to specific sequences in the 3′-untranslated region of target mRNAs. Members of this protein family share a conserved Puf domain consisting of eight 36 amino acid imperfect repeats. Here we report two Puf family member genes, PfPuf1 and PfPuf2, from the human malaria parasite Plasmodium falciparum. Both genes are spliced with four and three introns clustered within or near the Puf domains, respectively. Northern and RT–PCR analysis indicated that both genes were differentially expressed in gametocytes during erythrocytic development of the parasite. Except for similarities in the Puf domain and expression profile, the deduced PfPuf1 and PfPuf2 proteins differ considerably in size and structure. PfPuf1 has 1894 amino acids and a central Puf domain, whereas PfPuf2 is much smaller with a C-terminal Puf domain. The presence of at least two Puf members in other Plasmodium species suggests that these proteins play evolutionarily similar roles during parasite development. Both in vivo studies using the yeast three-hybrid system and in vitro binding assays using the recombinant Puf domain of PfPuf1 expressed in bacteria demonstrated intrinsic binding activity of the PfPuf1 Puf domain to the NRE sequences in the hunchback RNA, the target sequence for Drosophila Pumilio protein. Altogether, these results suggest that PfPufs might function during sexual differentiation and development in Plasmodium through a conserved mechanism of translational regulation of their target mRNAs.
PMCID: PMC135818  PMID: 12409450
22.  iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions 
PLoS Biology  2010;8(10):e1000530.
Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects.
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5′ splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5′ splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5′ splice sites. Surprisingly, TIA binding at 5′ splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3′ splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5′ splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.
Author Summary
Studies of splicing regulation have generally focused on RNA elements located close to alternative exons. Recently, it has been suggested that splicing of alternative exons can also be regulated by distal regulatory sites, but the underlying mechanism is not clear. The TIA proteins are key splicing regulators that enhance the recognition of 5′ splice sites, and their distal effects have remained unexplored so far. Here, we use a new method to map the positions of TIA-RNA interactions with high resolution on a transcriptome-wide scale. The identified binding positions successfully predict the local enhancing and distal silencing effects of TIA proteins. In particular, we show that TIA proteins can regulate distal alternative 3′ splice sites by binding at the 5′ splice site of the preceding exon. This result suggests that alternative splicing is affected by the timing of alternative exon definition relative to the recognition of the preceding 5′ splice site. These findings highlight the importance of analysing distal regulatory sites in order to fully understand the regulation of alternative splicing.
doi:10.1371/journal.pbio.1000530
PMCID: PMC2964331  PMID: 21048981
23.  Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA 
Background
Due to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. To further facilitate the application of the PUF domain for the sequence-specific RBP engineering, a rapid cloning approach is desirable that would allow efficient introduction of multiple key amino acid mutations in the protein. Here, we report the implementation of the Golden Gate cloning method for an efficient one-step assembly of a designer PUF domain for RNA specificity engineering.
Results
We created a repeat module library that is potentially capable of generating a PUF domain with any desired specificity. PUF domains with multiple repeat modifications for the recognition of altered RNA targets were obtained in a one-step assembly reaction, which was found to be highly efficient. The new PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular approach for PUF engineering. To demonstrate the application of the PUF domain assembly method for RBP engineering, we fused the PUF domain to a post-transcriptional regulator and observed a sequence-specific reporter and endogenous gene repression in human cell lines.
Conclusions
The Golden Gate based cloning approach thus should allow greater flexibility and speed in implementing the PUF protein scaffold for engineering designer RBPs, and facilitate its use as a tool in basic and applied biology and medicine.
doi:10.1186/1754-1611-8-7
PMCID: PMC3943411  PMID: 24581042
Protein engineering; RNA-binding protein; Post-transcriptional regulation; PUF; Pumilio; Tristetraprolin; TTP; Golden Gate
24.  The doublesex Splicing Enhancer Components Tra2 and Rbp1 Also Repress Splicing through an Intronic Silencer▿  
Molecular and Cellular Biology  2006;27(2):699-708.
The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.
doi:10.1128/MCB.01572-06
PMCID: PMC1800821  PMID: 17101798
25.  A Stochastic View of Spliceosome Assembly and Recycling in the Nucleus  
PLoS Computational Biology  2007;3(10):e201.
How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. To address this question, we compared the intranuclear trafficking kinetics of small nuclear ribonucleoprotein particles (snRNP) and non-snRNP proteins in the presence and absence of splicing activity. Photobleaching experiments clearly show that spliceosomal proteins move continuously throughout the entire nucleus independently of ongoing transcription or splicing. Using quantitative experimental data, a mathematical model was applied for spliceosome assembly and recycling in the nucleus. The model assumes that splicing proteins move by Brownian diffusion and interact stochastically with binding sites located at different subnuclear compartments. Inhibition of splicing, which reduces the number of pre-mRNA binding sites available for spliceosome assembly, was modeled as a decrease in the on-rate binding constant in the nucleoplasm. Simulation of microscopy experiments before and after splicing inhibition yielded results consistent with the experimental observations. Taken together, our data argue against the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent transcripts. Rather, the results suggest that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with pre-mRNAs.
Author Summary
Understanding the genomic program of an organism requires knowledge of how the information encoded in DNA is processed to generate messenger RNAs that can be translated into proteins. The initial products of gene transcription are extensively modified in the cell nucleus, and a major processing reaction consists of splicing of specific sequences from the middle of the primary transcripts. Splicing is catalyzed by the spliceosome, a large complex composed of five small RNAs and over 100 different proteins. Spliceosomes form anew on primary transcripts and disassemble after splicing, but what triggers the recruitment of individual spliceosomal components to selected gene products is unclear. Here, we have combined imaging and computational approaches to address this question. We obtained quantitative experimental data on the mobility and subnuclear distribution of splicing proteins before and after splicing inhibition, and we applied mathematical models to analyze and interpret the results. We conclude that spliceosomal components do not require a signal in order to be recruited to nascent transcripts. Our results favor the view that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with primary gene products.
doi:10.1371/journal.pcbi.0030201
PMCID: PMC2041977  PMID: 17967051

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