Mammalian Splicing Factor Research Articles

The Role of LUC7L2 in Splicing and MDS

Chromosomal abnormalities are common in myelodysplastic syndrome (MDS) and other myeloid malignancies. -7/del7q is found in 15% of MDS patients, but the pathological consequences of this deletion are unknown. To identify genes in the -7/del7q region that may contribute to pathogenesis of MDS, whole exome sequencing of MDS patient samples was used to detect novel somatic nonsense/frameshift mutations. Novel somatic mutations in the gene LUC7L2 were found in 27 malignant cases.Cases of mutant LUC7L2 hemizygosity, heterozygosity and homozygosity were observed in this cohort. The mutations were frameshift or nonsense, resulting in a premature stop codon and decreased expression of the LUC7L2 mRNA. LUC7L2 is located in the most commonly deleted region of -7/del7q; 7q34, which is deleted in 85% of -7/del7q patients. Patients with LUC7L2 mutations and those harboring -7/del7q have similar and statistically shorter overall survival than those with normal LUC7L2 expression levels.LUC7L2 is a poorly characterized, splicing-related protein. The function of LUC7L2 is largely unknown, but it is the ortholog of yeast protein LUC7p, which is involved in recruitment of early splicing factors. Recent studies have shown that over 65% of MDS patients harbor mutations in one of several proteins involved in pre-mRNA splicing. Therefore, we hypothesize that LUC7L2 acts as a mammalian splicing factor and deficiency of LUC7L2 results in aberrant splicing of transcripts that contribute to the pathogenesis of MDS.We characterized the role LUC7L2 in splicing by showing that LUC7L2 interacts with splicing factors using immunoprecipitation followed by mass spectrometry. Immunoprecipitation of LUC7L2 and interacting proteins revealed that LUC7L2 interacts with many proteins in the SR and HnRNP families of splicing regulators suggesting that LUC7L2 may also play a role as a splicing regulator. Splicing regulatory proteins are a diverse group that can influence constitutive splicing as well as alternative splicing. Therefore, to understand the mRNA targets of LUC7L2, we performed RNA CLIP-Seq. This revealed that LUC7L2 binds at least 301 pre-mRNA transcripts as well as spliceosomal snRNAs. LUC7L2 binding is enriched near splice junctions, with 117 of binding sites lying within 100bp of a splice site.To understand how LUC7L2 regulates splicing, we knocked down LUC7L2 in HEK293 cells and subjected the cells to a qRT-PCR based intron splicing efficiency assay as well as a splicing sensitive microarray. The intron splicing efficiency assay was performed on a subset of LUC7L2 targets identified by CLIP-Seq and showed that LUC7L2-deficiency often promotes the splicing of introns, suggesting that LUC7L2 acts as a splicing repressor. To understand the effect of LUC7L2 deficiency on all LUC7L2-targets, the splicing sensitive microarray identified splicing changes near LUC7L2-binding sites. LUC7L2-deficiency significantly alters the splicing of 151 transcripts that contain LUC7L2 binding sites.Missplicing of transcripts can alter protein function, leading to profound repercussions in the cell. The microarray analysis of LUC7L2-deficient cells identified several misspliced transcripts that are involved in the processes of proliferation, differentiation, and apoptosis, which are disrupted in MDS hematopoietic stem cells. Exons 2, 7 and 8 of the RUNX1 transcript are frequently skipped in the LUC7L2 knockdown cells. Although the mRNA expression of RUNX1 remains the same, several downstream transcripts that are regulated by RUNX1 have significantly increased or decreased expression, suggesting altered function of the RUNX1 protein. RUNX1 is a transcription factor that is required for HSC differentiation into myeloid progenitor cells. Altered splicing of RUNX1 and changes in the RUNX1 pathway could cause defects in the HSC differentiation process.In conclusion, novel somatic mutations in have been observed in LUC7L2 in patients with MDS. LUC7L2 is a protein that acts as a splicing regulator and deficiency of this protein results in missplicing of transcripts, including factors involved in differentiation, which may contribute to the pathogenesis of MDS. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding.

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification in mammalian messenger and non-coding RNAs. We report here that human METTL14 catalyzes m6A RNA methylation. Together with METTL3, the only previously known m6A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-14 that functions in cellular m6A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

The essential splicing factors U2AF⁶⁵ and SF1 cooperatively bind consensus sequences at the 3' end of introns. Phosphorylation of SF1 on a highly conserved "SPSP" motif enhances its interaction with U2AF⁶⁵ and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF⁶⁵/3' splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF⁶⁵ at 2.29 Å resolution and of the unphosphorylated SF1 domain at 2.48 Å resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF⁶⁵ interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full-length (P)SF1/U2AF⁶⁵/3' splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF⁶⁵ interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.

Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.

Interactome for auxiliary splicing factor U2AF 65 suggests diverse roles

U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kDa U2AF 35 (U2AF1) and the 65 kDa U2AF 65 (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF 65 as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF 65. Using a yeast two-hybrid system, we found fourteen U2AF 65-interacting proteins. The validity of the screen was confirmed by identification of five known U2AF 65-interacting proteins, including its heterodimeric partner, U2AF 35. In addition to binding these known partners, we found previously unrecognized U2AF 65 interactions with four splicing-related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF 65 homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF 65 each of these proteins interacts with and we discuss their potential roles in regulation of pre-mRNA splicing, 3′-end mRNA processing, and U2AF 65 sub-nuclear localization. These findings suggest expanded roles for U2AF 65 in both splicing and non-splicing functions.

Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors

A genome wide identification of mRNAs that were associated with the splicing factor subunit U2AF65 suggests that U2AF65 associates with specific subsets of spliced mRNAs and may be involved in novel cellular functions in addition to splicing.

Shigella effector IpaH9.8 binds to a splicing factor U2AF 35 to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, ΔipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF 35, a mammalian splicing factor, which interferes with U2AF 35-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF 35 level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1β genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.

Tau Exons 2 and 10, Which Are Misregulated in Neurodegenerative Diseases, Are Partly Regulated by Silencers Which Bind a SRp30c·SRp55 Complex That Either Recruits or Antagonizes htra2β1

Tau Exons 2 and 10, Which Are Misregulated in Neurodegenerative Diseases, Are Partly Regulated by Silencers Which Bind a SRp30c·SRp55 Complex That Either Recruits or Antagonizes htra2β1

A Pathway of Sequential Arginine-Serine-Rich Domain-Splicing Signal Interactions during Mammalian Spliceosome Assembly

Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF 65 and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5′ splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.

Strange bedfellows: polyadenylation factors at the promoter.

One of the more unexpected discoveries in the field of gene expression during the last few years has been the degree to which transcription of mRNA precursors is coupled to their subsequent processing into mature mRNAs. Much of this linkage involves the transcribing enzyme itself, RNA polymerase II (RNAP II), and specifically the repetitive C-terminal domain of its largest subunit (known simply as the CTD). A large body of evidence now indicates that the CTD has a significant role in enhancing the efficiency of all three of the major processing reactions—capping, splicing, and polyadenylation (for review, see Hirose and Manley 2000; Bentley 2002; Maniatis and Reed 2002; Proudfoot et al. 2002). This was initially surprising—all three reactions were known to occur just fine in vitro in the absence of transcription, or for that matter RNAP II and the CTD—but in retrospect it makes sense as a way to ensure accurate, efficient, and rapid processing of nascent pre-mRNAs. Precisely how the CTD functions is not entirely understood, especially in the more complex splicing and polyadenylation reactions, but a number of interactions with specific processing factors have been documented and these likely serve to help recruit the processing machinery to the pre-mRNA and then to stabilize or enhance the activity of these complexes. Interactions between the transcription and processing machineries extend beyond those by which RNAP II and the CTD stimulate processing (for review, see Manley 2002). For example, yeast capping enzymes seem able to repress transcription reinitiation, perhaps as a checkpoint to ensure that capping has been completed (Myers et al. 2002), whereas mammalian splicing factors can function in vitro to stimulate transcription initiation (Kwek et al. 2002) or elongation (Fong and Zhou 2001). In this brief review, we discuss evidence supporting the existence of a complex, evolutionarily conserved set of interactions that link even more tightly events that occur at the promoter to define the transcription start site and mRNA 5 end with those that occur at the other end of the gene to produce the mRNA 3 end and ultimately the transcription stop site.

CDNA cloning and mRNA expression of Transformer 2 (Tra 2) in chicken embryo

We report the cloning of a chicken Transformer 2 (Tra 2) cDNA that encodes a protein of 289 amino acids which are 97.9% identical to those of mammalian splicing factor, Tra 2. Tra 2 mRNA was expressed in chicken embryonic tissues and was observed as a band of 1.5 kb by Northern blot analysis. Whole mount in situ hybridization showed an mRNA expression of Tra 2 in telencephalon, mandible, hyoid arch, wing and leg buds as early as day 3.5 of incubation. These results suggest that the Tra 2 gene may play a role in organogenesis in the chicken embryo.

Characterization of U2AF(6), a splicing factor related to U2AF(35).

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.

Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex.

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative alpha helices, a highly conserved loop, and parts of a beta sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323-332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites.

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon.

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.

ZFM1/SF1 mRNA in rat and gerbil brain after global ischaemia.

Cerebral ischaemia results in significant brain damage, but the molecular mechanisms associated with ischaemia-induced brain injury are not well defined. We have adopted an improved differential-display method to search for new ischaemia-related genes. Among the different cDNAs isolated following transient forebrain ischaemia in rat, PH3.3 was selected for further studies. The search for homologies revealed that it is the rat homologue to human zinc finger motif 1 (ZFM1), also called mammalian splicing factor 1 (SF1). With Northern blot, PH3.3 hybridized with three mRNA species of 2.3, 2.9 and 3.6 kb, significantly increased at 6 h and 5 days after the ischaemic insult. These findings were extended also to another animal model. In situ hybridization in ischaemic gerbils showed that PH3.3 mRNA was induced in the dentate gyrus as early as 4 h post-ischaemia. Expression peaked at 2 days in the whole hippocampus and cortex, and then progressively decreased towards sham levels. By day 4, expression had disappeared almost entirely from the cells in the CA1 region of the hippocampus, concomitant with the degeneration of pyramidal neurons. Interestingly, ZFM1/SF1 has been recently identified as activated following p53-induced apoptosis. Several lines of evidence suggest that p53 may play two roles in the post-ischaemic brain. The primary role of p53 is to activate DNA repair processes, but if repair fails, apoptosis will be initiated. Thus, ZFM1/SF1 may represent a relevant link between p53 and the neuroprotective/neurodegenerative processes which follow cerebral ischaemia.

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors.

The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF/SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.

A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition.

During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts. Recently, we identified a branchpoint sequence binding protein in yeast (BBP). The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract. In this paper we demonstrate that the mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and we suggest that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing. We also demonstrate the essential nature of the third RBD of U2AF65 for the interaction between the two proteins, both in the presence and absence of RNA.

The Splicing Factor BBP Interacts Specifically with the Pre-mRNA Branchpoint Sequence UACUAAC

The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.