Splice Site Signals Research Articles

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes.

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.

Human U2AF65 Recognizing the Major Polypyrimidine Splice Site Signal

Disease-causing mutations often occur in the polypyrimidine (Py) tract splice site signals of human gene transcripts. The essential U2AF65 protein recognizes Py tracts near 3´ splice sites and initiates assembly of the splicing "machine", a megadalton complex comprised of approximately 100 proteins and five small nuclear RNAs. Our prior structures of a shortened U2AF65 variant reveal the basis for nucleotide interactions at subset of binding sites. How intact U2AF65 recognizes the Py tract splice site signal remains unknown to date. We determined a 2.0 Å resolution structure of intact U2AF65 recognizing an optimal, all-uridine Py tract. The new structure and complementary biochemical experiments reveal integral roles for main-chain atoms of the interdomain linker and residues surrounding two core RNA recognition motifs (RRM1 and RRM2) in recognition of the Py tract. The new U2AF65 structural information sheds light on the splicing defects caused by Py tract mutations in human genetic diseases. We test the U2AF65 structural relationship for a representative Py mutation that causes X-linked retinitis pigmentosa.

Somatic spliceosomal factor mutations in bone marrow neoplasms ő downstream effects and mechanistic insights (560.3)

Somatic mutations of spliceosomal proteins such as U2AF1, SF3B1, SRSF2 and PRPF8 have been identified in myeloid malignancies. These mutations are usually single heterozygous missense mutations at highly recurrent sites. While all of these mutations affect proteins in the common splicing pathway, their downstream consequences may be diverse and involve distinct oncogenic pathways. To identify affected genes and gain mechanistic insight into the effects of these mutations, we have analyzed the tumor transcriptomes of multiple AML patients with and without spliceosomal factor mutations. For U2AF1, we analyzed 6 tumors with U2AF1 mutations and 14 tumors that had no known splicing factor mutations. Focusing on cases of alternative exon inclusion, we identified 35 exons in 35 genes whose inclusion was altered using stringent statistical cutoffs. Of these, 8 exons were more included while 27 were more excluded in the U2AF1 mutant tumors. We examined the splice site signals flanking the alternative exons and noted a highly unusual sequence pattern adjacent to the 3’ AG dinucleotide, which is the recognition site of U2AF1. The sequence at this position showed a mutually exclusive pattern upon comparing the excluded to the included exons and this pattern also differed from the consensus 3’ splice site sequence. Biochemical analysis of the RNA binding specificities of purified mutant and wild type U2AF1/U2AF2 heterodimers showed alterations that correlated with the splicing alterations observed in vivo. Engineered cell lines expressing the mutant U2AF1 protein recapitulated the splicing alterations observed in vivo. These results suggest that the mutations in U2AF1 lead to altered splice site recognition and aberrant splicing of a subset of genes that contribute to the neoplastic state. Similar analyses of the splicing effects of mutations in other spliceosomal factors showed that there is little overlap in the most affected genes.Grant Funding Source: Supported by NIH grant HL118281

Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

BackgroundGenome-wide computational analysis of alternative splicing (AS) in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs.ResultsOur analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy.ConclusionsThe extent of AS in Chlamydomonas that we observed is much smaller than observed in land plants, but is much higher than in simple unicellular heterotrophic eukaryotes. The percentage of different alternative splicing events is similar to flowering plants. Prevalence of constitutive and alternative splicing in Chlamydomonas, together with its simplicity, many available public resources, and well developed genetic and molecular tools for this organism make it an excellent model system to elucidate the mechanisms involved in regulated splicing in photosynthetic eukaryotes.

Thermodynamic Characteristics of pre-mRNA Splice Site Recognition

Here, we reveal that an unusually large enthalpy-entropy compensation underlies recognition of polypyrimidine (Py) splice site signals. Competitive binding to Py tract splice site signals represents a prevalent means for alternative pre-mRNA splicing. The thermodynamic forces driving association of splicing factors with single-stranded (ss) pre-mRNAs represents a gap in the current understanding of splice site selection. We compared Py tract interactions among three splicing factors: (1) U2AF65, an essential pre-mRNA splicing factor that recognizes constitutive 3′ splice site signals; (2) Sex-Lethal, a prototypical alternative splicing factor that antagonizes U2AF65; and (3) TIA-1, an alternative splicing factor that promotes use of specific 5′ splice sites. All three proteins bound polyuridine (U20) sequences with comparable or higher affinity than natural splice site sequences in fluorescence anisotropy assays. Consistent with the ability of Sex-Lethal to outcompete U2AF65 during splice site selection, U2AF65 displayed the lowest and Sex-Lethal the highest affinities for the RNA sites.The enthalpic and entropic contributions were investigated in detail using ITC, initially using the homogeneous U20 site to avoid sequence-dependent complications of the binding isotherms. All three splicing factors exhibited an unusually large enthalpy-entropy compensation underlying U20 binding, with magnitudes ∼10-fold greater than those of typical protein-protein or protein-ligand complexes. Given that full thermodynamic characterizations of protein association with single-stranded RNAs are rare, this raised the question of the source of this unusual thermodynamic signature: Is a large enthalpy-entropy compensation a general characteristic of ssRNA binding, an inherent property of Py tracts, or a signature of sequence-specific ssRNA recognition? These possibilities are clarified by thermodynamic comparison of purine-tract association by these Py tract splicing factors, contrasted with the purine-specific protein PAB.

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing

Studies of expressed sequence tag data sets have revealed large numbers of splicing variants for human genes, but it remains challenging to distinguish functionally important variants from aberrant splicing, clarify the nature of the alternative functions, and understand the signals that regulate splicing choices. To help address these issues, we have constructed and analyzed a large data set of 1,478 exon-skipping alternative splicing (AS) variants evolutionarily conserved in human and mouse. In about one-fifth of cases, one isoform appears subject to nonsense-mediated mRNA decay (NMD), supporting the idea that a major role of AS is to regulate gene expression; one-quarter of these NMD-inducing cases involve a conserved exon whose apparent sole purpose is to mediate destruction of the message when included. We explore sequence conservation likely related to splicing regulation, using in part a measure of the overall amount of conserved information in a sequence, and find that the increased conservation that has been observed within AS exons primarily affects synonymous sites, suggesting that regulatory signals significantly constrain synonymous substitution rates. We show that a lower frequency of the inclusion isoform relative to the exclusion isoform tends to be associated with weaker splice site signals, smaller exon size, and higher intronic sequence conservation, and provide evidence that all of these factors are under selection to control relative isoform frequencies. Some conserved instances of AS appear to represent aberrant splicing events that by chance have occurred in both species, and we develop a nonparametric likelihood approach to identify these.

Variation in sequence and organization of splicing regulatory elements in vertebrate genes

Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.

Relative exon affinities and suboptimal splice site signals lead to non-equivalence of two cassette exons.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exons 2 and 3 of the gene are alternatively spliced cassettes in which exon 3 never appears independently of exon 2. Expression of tau minigene constructs in cells indicate that exon 2 resembles a constitutive exon, while a suboptimal branch point connected to exon 3 inhibits inclusion of exon 3 in the mRNA. Splicing of the two tau exons is controlled by their relative affinities for each other versus the affinities of their flanking exons for them.

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.

Categorical discriminant analysis of 3′-splice site signals of mRNA precursors in higher eukaryote genes

With regard to the signals that direct excision of introns from mRNA precursors in higher eukaryote genes, a consensus sequence, (sequence; see text); has been proposed for the 3'-splice site, but actual 3'-splice site sequences differ from it to a greater or lesser degree. In the present paper, nucleotide sequences were transformed into categorical data, and quantification analysis (class II), as proposed by Hayashi, was applied to the system. Categorical weights given to variables related to position and the species of nucleotide were estimated so that the two classes of 3'-splice site sequences and sequences other than 3'-splice site might be discriminated most distinctly. The 3'-splice site signals were then characterized in terms of these categorical weight values. We also calculated partial correlation coefficient values, which explain the relative importance of each position in the 3'-splice site signal sequence.

The α- and β-tubulins of Toxoplasma gondii are encoded by single copy genes containing multiple introns

We have studied the fundamentals of gene expression in the protozoan parasite Toxoplasma gondii by analyzing, in detail, the genes encoding α- and β-tubulin. Southern analysis and quantitation studies reveal that, unlike in other organisms studied thus far, both these genes are present as single copies in the haploid Toxoplasma genome. Sequencing of these genes indicates that they both contain multiple introns with conserved 5′ and 3′ splice site signals. We have found that HeLa cell nuclear extracts are able to accurately splice a Toxoplasma pre-mRNA construct. We have mapped, for the α-tubulin gene, the exact site of transcription initiation and the approximate site of poly A addition by primer extension and RNase protection assays. Trans-splicing, as demonstrated in the Kinetoplastida, is not involved in the formation of the mature α-tubulin transcript in T. gondii.

DNA sequences and multivariate statistical analysis. Categorical discrimination approach to 5’ splice site signals of mRNA precursors in higher eukaryotes' genes

The signals which direct excision of introns from mRNA precursors in higher eukaryotes' genes are not well understood. Although a consensus sequence, CAAG/GTAGAGT, has been proposed with the 5' splice site, actual 5' splice site sequences differ from it to a greater or lesser degree. In order to study such a signal more quantitatively, nucleotide sequences were transformed into categorical data, and multivariate statistical analysis was applied to such a system. Categorical weights on the variables were estimated in such a way that the two classes of 5' splice site sequences and sequences other than 5' splice site might be discriminated most distinctly. The 5' splice site signals were then characterized in terms of those statistical results.

Splice-Site Signals of mRNA Precursors as Revealed by Computer Search. Site-Specific Mutagenesis and Thalassemia

Many eukaryotic genes are interrupted by introns, which are removed from mRNA precursors by the RNA splicing mechanism. Although nucleotide sequences around splice sites have considerable homology among various genes, exact splice-site signals are unknown. Previously, applying the computer searching method to primary nucleotide sequences of various genes, we studied what kinds of patterns are the necessary and sufficient sequences for recognition of the 5'-slice site (donor site). We proposed that four common patterns, AG/GTA, /GTAAGT, RG/GTGAG, and AG/GTXXGT, where R = A or G and X = A, T, G, or C, are often used as such signals. In the present paper, we examined a number of experimental results on site-specific mutagenesis around donor sites and on alpha- and beta-thalassemia in globin genes, and found that the above four common patterns could explain almost all of those results.