Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP.

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5'' splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5'' splice site and by an exonic enhancer differ mechanistically.     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

Determinants of plant U12-dependent intron splicing efficiency

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated.     

Branchpoint selection in the splicing of U12-dependent introns in vitro

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.     

Role of the 3' splice site in U12-dependent intron splicing

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.     

A novel splicing regulator shares a nuclear import pathway with SR proteins

Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.     

Domains of human U4atac snRNA required for U12-dependent splicing in vivo

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5' stem-loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3' stem-loop element, were active for splicing. Complete deletion of the 3' stem-loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction.     

Regulation of splicing by SR proteins and SR protein-specific kinases

Genomic sequencing reveals similar but limited numbers of protein-coding genes in different genomes, which begs the question of how organismal diversities are generated. Alternative pre-mRNA splicing, a widespread phenomenon in higher eukaryotic genomes, is thought to provide a mechanism to increase the complexity of the proteome and introduce additional layers for regulating gene expression in different cell types and during development. Among a large number of factors implicated in the splicing regulation are the SR protein family of splicing factors and SR protein-specific kinases. Here, we summarize the rules for SR proteins to function as splicing regulators, which depend on where they bind in exons versus intronic regions, on alternative exons versus flanking competing exons, and on cooperative as well as competitive binding between different SR protein family members on many of those locations. We review the importance of cycles of SR protein phosphorylation/dephosphorylation in the splicing reaction with emphasis on the recent molecular insight into the role of SR protein phosphorylation in early steps of spliceosome assembly. Finally, we highlight recent discoveries of SR protein-specific kinases in transducing growth signals to regulate alternative splicing in the nucleus and the connection of both SR proteins and SR protein kinases to human diseases, particularly cancer.     

Lsm proteins promote regeneration of pre-mRNA splicing activity

Lsm proteins are ubiquitous, multifunctional proteins that affect the processing of most RNAs in eukaryotic cells, but their function is unknown. A complex of seven Lsm proteins, Lsm2-8, associates with the U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs. Spliceosomes contain five snRNAs, U1, U2, U4, U5, and U6, that are packaged as ribonucleoprotein particles (snRNPs). U4 and U6 snRNAs contain extensive sequence complementarity and interact to form U4/U6 di-snRNPs. U4/U6 di-snRNPs associate with U5 snRNPs to form U4/U6.U5 tri-snRNPs prior to spliceosome assembly. Within spliceosomes, disruption of base-paired U4/U6 heterodimer allows U6 snRNA to form part of the catalytic center. Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, although little is known about this process. Here we present evidence that regeneration of splicing activity in vitro is dependent on Lsm proteins. RNP reconstitution experiments with exogenous U6 RNA show that Lsm proteins promote the formation of U6-containing complexes and suggest that Lsm proteins have a chaperone-like function, supporting the assembly or remodeling of RNP complexes involved in splicing. Such a function could explain the involvement of Lsm proteins in a wide variety of RNA processing pathways.     

Thermodynamic analyses of the constitutive splicing pathway for ovomucoid pre-mRNA

The ovomucoid pre-mRNA has been folded into mini-hairpins adaptable for the RNA recognition motif (RRM) protein binding. The number of mini-hairpins were 372 for pre-mRNA and 83-86 for mature m RNA The spatial arrangements are, in average, 16 nucleotides per mini-hairpin which includes 7 nt in the stem, 5.6 nt in the loop and 3.7 nt in the inter-hairpin spacer. The constitutive splicing system of ovomucoid-pre-mRNA is characterized by preferred order of intron removal of 5/6 > 7/4 > 2/1 > 3. The 5′ splice sites (5′SS), branch point sequences (BPS) and 3′ splice sites (3′SS) were identified and free energies involved have been estimated in 7 splice sites. Thermodynamic barriers for splice sites from the least (| lowest | -Kcal) were 5,4, 7,6, 2,1, and 3; i.e., −18.7 Kcal, −20.2 Kcal, −21.0 Kcal, −24.0 Kcal, −25.4 Kcal, −26.4 Kcal and −28.2 Kcal respectively. These are parallel to the kinetic data of splicing order reported in the literature. As a result, the preferred order of intron removals can be described by a consideration of free energy changes involved in the spliceosomal assembly pathway. This finding is consistent with the validity of hnRNP formation mechanisms in previous reports.     

Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.     

The role of U5 snRNP in pre-mRNA splicing.

The current model for the function of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome proposes that U5 carries binding sites for the 5' and 3' exons, allowing the spliceosome to 'tether' the 5' exon intermediate produced by the first catalytic step and align it with the 3' exon for the second step. Functional analysis of U5 snRNA in cis-spliceosomes has provided support for this model, and data from nematode and trypanosome splicing systems suggest that U5 or a U5-like snRNA performs a similar role in trans-splicing.     

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.     

Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.     

Shuttling SR proteins: more than splicing factors

Serine-arginine (SR) proteins commonly designate a family of eukaryotic RNA binding proteins containing a protein domain composed of several repeats of the arginine-serine dipeptide, termed the arginine-serine (RS) domain. This protein family is involved in essential nuclear processes such as constitutive and alternative splicing of mRNA precursors. Besides participating in crucial activities in the nuclear compartment, several SR proteins are able to shuttle between the nucleus and the cytoplasm and to exert regulatory functions in the latter compartment. This review aims at discussing the properties of shuttling SR proteins with particular emphasis on their nucleo-cytoplasmic traffic and their cytoplasmic functions. Indeed, recent findings have unravelled the complex regulation of SR protein nucleo-cytoplasmic distribution and the diversity of cytoplasmic mechanisms in which these proteins are involved.