Conservation of functional domains involved in RNA binding and protein–protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1

PUF60 is an essential splicing factor functionally related and homologous to U2AF65 . Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophancontaining linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 Å resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF 65 , and SF3b155. Compared with U2AF 65 -UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF 65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.Pre-mRNA splicing is a stepwise process initiated by the recognition of sequence elements at the splice site by specific splicing factors (1). The branch point sequence is recognized by splicing factor SF1 (2, 3), whereas the polypyrimidine tract and the 3Ј splice site AG-dinucleotide are bound by the heterodimer U2AF 65 -U2AF 35 (4 -7). Although SF1 alone interacts only weakly with the branch point sequence, this interaction is stabilized significantly by U2AF 65 , which binds simultaneously to SF1 and to the polypyrimidine tract (8). In the next step of splicing initiation U2 snRNP is brought to the 3Ј splice site. This involves base pairing of the U2 RNA to the branch site RNA (9) and localization of the SF3b subunit p14 near the branch point adenosine by an interaction with the N terminus of the U2 snRNP component . Initial contacts between the U2 snRNP and the pre-mRNA are mediated by the N terminus of SF3b155 binding to U2AF 65 and displacing SF1 from the branch point sequence (13).The -UHM/ SF1-ULM (15), and the UHM of SPF45 (splicing factor 45 kDa) bound to an ULM in SF3b155 (23) all share a very similar mode of molecular recognition, suggesting that UHMs in other proteins might bind similar linear motifs as well. Because the ULM consensus motif is rather short ((K/R) 4 -6 X 0 -1

mentioning

confidence: 99%

Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60

Corsini

Hothorn

Stier

et al. 2009

“…1A). (i) As addressed here, two RNA recognition motifs (RRMs) recognize the Py tract splice site signal (11,12); (ii) a region near the RRMs recruits the ATPase UAP56 to the assembling spliceosome (13); (iii) a C-terminal U2AF homology motif (UHM) domain organizes the SF1 (14) and SF3b155 (15) splicing factors at the branch point sequence; (iv) a tryptophan-containing UHM ligand motif (16) positions the U2AF 35 small subunit at the 3Ј splice site (17); and (v) an N-terminal arginine-serine-rich (RS) domain promotes branch point sequence/U2 small nuclear RNA annealing (18). To accomplish these tasks, both the C-terminal UHM of U2AF 65 and the N-terminal RS domain are required to interact with splicing factors and RNA sites located upstream (5Ј), respectively, of the Py tract binding site.…”

mentioning

confidence: 99%

Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65

Jenkins

Shen

Green

et al. 2008

The U2 auxiliary factor large subunit (U2AF 65 ) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem Pre-mRNA splicing is an essential source of transcript diversity in multicellular eukaryotes (reviewed in Refs. 1 and 2), as reflected by the significant number of cancers and hereditary diseases associated with mutations in pre-mRNA splice site signals or splicing factors (3-5). The splicing machinery (spliceosome) is faced with the task of recognizing relatively short exons (ϳ150 nucleotides on average in the human genome) located within vast stretches of intron RNA (Ͼ1500 nucleotides) (6). Although consensus sequences mark the 5Ј and 3Ј splice sites of the pre-mRNA, these sequences are relatively short and degenerate so that cryptic splice sites outnumber bona fide splice sites by an order of magnitude (7). Furthermore, to be distinguished and regulated in a specific manner, "weak" alternative splice sites may deviate substantially from the optimal consensus of "strong," constitutive splice sites (8). The consensus sequences of 3Ј splice sites recognized by the major spliceosome are more extensive than those of 5Ј splice sites (9) and include a branch point sequence closely followed by a polypyrimidine (Py) 4 tract, which is primarily composed of uridines and cytidines. How the spliceosome accurately identifies and pairs the splice sites remains an outstanding question.Assembly of the core spliceosome (U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles) first requires splice site identification by the U2 auxiliary factor large subunit (U2AF 65 ) (10). The U2AF 65 subunit serves as an extensive molecular surface, with domains responsible for critical functions (Fig. 1A). (i) As addressed here, two RNA recognition motifs (RRMs) recognize the Py tract splice site signal (11, 12); (ii) a region near the RRMs recruits the ATPase UAP56 to the assembling spliceosome (13); (iii) a C-terminal U2AF homology motif (UHM) domain organizes the SF1 (14) and SF3b155 (15) splicing factors at the branch point sequence; (iv) a tryptophan-containing UHM ligand motif (16) positions the U2AF 35 small subunit at the 3Ј splice site (17); and (v) an N-terminal arginine-serine-rich (RS) domain promotes branch point sequence/U2 small nuclear RNA annealing (18). To accomplish these tasks, both the C-terminal UHM of U2AF 65 and the N-terminal RS domain are required to interact with splicing factors and RNA sites located upstream (5Ј), respectively, of the Py tract binding site. Simultaneously, the UHM ligand motif near the U2AF 65 N terminus interacts with U2AF 35 bound to the 3Ј splice site. Directed hydroxyl radical footprinting shows that the U2AF 65 N terminus contacts pre-mRNA sequences

“…1). The BBP/SF1 N-terminal domain interacts with Mud2p in yeast and U2AF65 in humans (5,6,8). The C terminus contains a proline-rich domain, which in yeast interacts with Prp40, a component of the U1 snRNP (7).…”

mentioning

confidence: 99%

“…1). The region of BBP/SF1 responsible for binding the BPS contains a heterogeneous nuclear ribonucleoprotein K homology (KH) domain, a QUA2 domain, and one or two zinc knuckles (1,6,12). Most KH proteins are RNA-binding proteins that have multiple KH domains arranged in multiple repeats (13-16).…”

mentioning

confidence: 99%

See 1 more Smart Citation

An Extended RNA Binding Site for the Yeast Branch Point-binding Protein and the Role of Its Zinc Knuckle Domains in RNA Binding

Garrey¹,

Voelker²,

Berglund

2006

The highly conserved branch point sequence (BPS) of UAC-UAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.The branch point-binding protein (BBP) 4 in Saccharomyces cerevisiae is an RNA-binding protein that is thought to be the first protein to bind the branch point sequence (BPS) during splicing. In yeast BBP binds to the highly invariant branch point sequence UACUAAC (branch point A is underlined), recognizing every nucleotide (1). BBP binds the BPS in the second commitment complex (CC2) and is necessary for stable formation of this complex (2). Splicing factor 1 (SF1), the human orthologue of BBP, is a more promiscuous RNA-binding protein, recognizing a degenerate sequence, CURAY (R ϭ purine, Y ϭ pyrimidine), in which only the U and A are critical (1). When discussing the yeast branch point-binding protein we will use the term BBP, when discussing the human protein we will use the term SF1, and when discussing both proteins we will use the term BBP/SF1. In the mammalian system, the equivalent complex to the yeast CC2 is the E complex in which SF1 binds the BPS (3, 4). In addition to recognizing the BPS, BBP/SF1 plays an important role in bridging the 5Ј end and 3Ј end of the intron by interacting with U1 snRNP at the 5Ј splice site and Mud2p at the 3Ј end of yeast introns and U2AF65 at the 3Ј end of human introns (5-8). BBP/SF1 is replaced at the BPS by U2 snRNP in an ATP-dependent step that leads to the formation of A complex (9, 10).BBP/SF1 contains multiple domains, many of which are conserved between yeast and human (Fig. 1). The BBP/SF1 N-terminal domain interacts with Mud2p in yeast and U2AF65 in humans (5,6,8). The C terminus contains a proline-rich domain, which in yeast interacts with Prp40, a component of the U1 snRNP (7). FBP11, a potential vertebrate homologue of Prp40, binds to the proline-rich domain of human SF1 (11).One of the most highly conserved regions of BBP/SF1 encompasses the RNA binding domains (Fig. 1). The region of BBP/SF1 responsible...