A Targeted Bypass Screen Identifies Ynl187p,Prp42p,Snu71p,and Cbp80p for Stable U1 snRNP/Pre-mRNA Interaction

To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5′ splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5′ splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5′ splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.Nuclear precursor mRNA (pre-mRNA) splicing takes place in the spliceosome, a large dynamic complex consisting of over 100 proteins and five small nuclear RNAs (snRNAs) (32, 70). During spliceosome assembly, the U1 small nuclear ribonucleoprotein particle (snRNP) first contacts the pre-mRNA 5′ splice site (5′ss), followed by binding of the U2 snRNP to the branch site and the joining of the U5-U4/U6 tri-snRNP (32, 64, 70). The step in which U1 snRNP binds to the 5′ss is arguably one of the most critical, because it probably commits pre-mRNA to the splicing pathway (38, 48, 49, 60, 74). In the budding yeast Saccharomyces cerevisiae in vitro system, two U1-snRNP-containing commitment complexes (CCs), CC1 and CC2, can be detected by native gel electrophoresis prior to the U2 snRNP''s joining to form the prespliceosome (38, 60). CC1, whose formation is dependent on a functional 5′ss, appears to be a kinetic precursor to CC2, whose formation requires both a functional 5′ss and branch site and the participation of the branch-site-binding protein (BBP) and Mud2p, which are likely equivalent to SF1 and U2AF65, respectively, in the mammalian system (1-3, 75).Accumulating evidence suggests that formation of the canonical 5- to 7-bp RNA duplex between U1 snRNA and the 5′ss region is not sufficient to cause a stable CC to form in the yeast system (59, 62, 78); protein-RNA contacts are also important. For example, Zhang and Rosbash (77) identified eight proteins, all present in CCs, that make physical contact with the pre-mRNA at or near the 5′ss. Four of these proteins, U1C, U1-70K, Snu56p, and Nam8p, are integral parts of the U1 snRNP (20), and another three, SmB, SmD1, and SmD3, belong to the seven-member ring that binds the conserved Sm site present on U1, U2, U4, and U5 snRNAs (33, 71). The remaining protein, Cbp80p, is a subunit of the nuclear cap-binding complex (CBC), which also contains a second subunit, Cbp20p (28, 39). Interestingly, despite being a non-snRNP factor, Cbp80p is known to collaborate with U1 snRNP to help form or stabilize CC1 (8, 40). Furthermore, the contact between the C-terminal tails of SmB, SmD1, and SmD3 and the pre-mRNA may contribute to stabilizing the U1 snRNP/pre-mRNA interaction (76). Finally, Du and Rosbash (11) more recently showed that U1C is capable of selecting splice-site-like sequences in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast splice-site consensus sequence.Once fully assembled, the spliceosome must progress through a number of major structural and conformational changes to form the catalytic center; these include a series of highly orchestrated RNA-RNA rearrangements (53, 64, 70). Some of these are mutually exclusive; i.e., the formation of one RNA duplex requires the disruption of another. For example, the base-pairing interaction between the U1 snRNA and the 5′ss is replaced by a U6 snRNA/5′ss pairing. This exchange appears to be coupled to U4/U6 RNA unwinding (53, 64, 70). It is now known that splicing factors belonging to the ATPase II superfamily (18), which are also termed the DEXD/H-box proteins (5, 43), promote spliceosomal RNA rearrangements (64). However, the precise roles of most DEXD/H-box proteins remain unclear.It has been nearly 2 decades since DEXD/H-box proteins were first proposed to be RNA helicases (44). Over the years, a wealth of data revealed that DEXD/H-box proteins are essential in most, if not all, RNA-related pathways, e.g., splicing, mRNA export, and ribosomal biogenesis (5, 43, 64). Their modes of action in vivo remain a mystery, however. For example, Lorsch and Herschlag (45, 46) proposed that DEXD/H-box proteins may perform functions which are distinct from RNA unwinding and include mediating large-scale RNA structural rearrangements, disrupting protein-RNA or protein-protein interactions, and functioning as fidelity sensors in RNA-RNA interactions and rearrangements. Indeed, recent data confirm that DEXD/H-box proteins can catalyze protein displacement in a manner independent of RNA duplex unwinding (30). Therefore, the essential functions of DExH/D proteins can be exerted on a wide range of RNP substrates. This “RNPase” (or ATPase for RNP remodeling) hypothesis appears especially attractive in light of the fact that RNA duplexes in vivo are rarely more than ∼10 contiguous base pairs in length and that they often require protein binding for stabilization (21, 63). To fully understand how DEXD/H-box proteins function in the cell, it is critical to identify their physiological substrates.Inspired by our previous finding that the requirement for Prp28p, an essential DEXD/H-box splicing factor, can be bypassed by mutations that alter the YHC1 gene, which encodes U1C protein (7), we sought to exploit the bypass concept to deepen our understanding of the role of Prp28p in splicing. The underlying hypothesis is that bypass mutations define gene products that Prp28p may counteract. Here we describe the outcome of this approach and provide a detailed analysis of Ynl187p, a novel protein that probably contributes to stabilizing the U1 snRNP-5′ss interaction.     

Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of Trypanosoma brucei

The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B″ protein from Trypanosoma brucei. U2B″ interacts with a second U2 snRNP protein, U2-40K (U2A′), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.In trypanosomes, trans-splicing is an essential step in the expression of all protein-coding genes. The resulting mRNAs always carry a noncoding spliced leader (SL) sequence of 39 nucleotides at their 5′ ends, which is derived from the SL RNA. In addition to the SL RNA, the small nuclear RNAs (snRNAs) U2, U4, U5, and U6 are essential cofactors during trans-splicing (reviewed in reference 14).In previous studies, we characterized some of the protein components of the spliceosomal small nuclear ribonucleoproteins (snRNPs) from Trypanosoma brucei. All snRNPs contain a core of seven Sm polypeptides (18). Recently, we reported that the identity of the Sm proteins varies among spliceosomal snRNPs; specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K (34). There is a similar case of Sm core variation in the U4 snRNP, where a single Sm protein, SmD3, is replaced by a U4-specific LSm protein (32; N. Jaé and A. Bindereif, unpublished data). Trypanosomal snRNAs also differ significantly from what we know in other systems, reflecting the large evolutionary distance and trypanosome-specific properties. For example, both the U1 and U5 snRNAs from trypanosomes represent the shortest known orthologues (6, 19).In addition to the Sm proteins, some snRNP-specific protein factors were found in trypanosomes. Sequence comparisons identified the U2-40K protein as the trypanosomal homologue of the human U2A′ protein (5), a finding that was unexpected, since no immunological relationship could be detected between these proteins (17). As characterized in other systems, including those of humans, yeasts, and plants (9, 29, 31), the U2 snRNP contains a second specific protein, U2B″, a protein closely related to the U1 snRNP protein U1A. Except for the Saccharomyces cerevisiae orthologue, the known U2B″ proteins are built of two RNA recognition motifs (RRMs), with the N-terminal RRM being responsible for snRNA binding specificity (25). The close relatedness of these two proteins is also reflected in Drosophila melanogaster, where a single protein, SNF/D25, combines the functions of both individual proteins (11, 22). Furthermore, genetic and functional redundancy was demonstrated for the two proteins in Caenorhabditis elegans (24).From previous studies on the mammalian U2 snRNP, we know that the U2- specific proteins U2A′ and U2B″ interact with each other, independently of U2 snRNA; moreover, U2B″ binds directly to loop nucleotides of stem-loop IV, but only with the assistance of interacting U2A′ (23, 25, 26). Analogous to the cis-splicing mechanism, the U2 snRNP is likely to play an important role in early trans-spliceosome assembly. Compared with the other snRNAs, the trypanosomatid U2 snRNA differs in several important aspects from its highly conserved counterparts in other species. First, stem-loop III is precisely deleted. Second, the branch point recognition region located between stem-loops I and IIa is missing; in parallel, there is no stringent consensus of branch points in the 3′-splice site region of the polycistronic pre-mRNA. Third, the Sm protein binding site does not follow the general consensus. Finally, only some of the otherwise highly conserved loop IV nucleotides occur in the trypanosomatid U2 snRNAs (7, 12, 15, 33).Here we report the identification and characterization of the U2-specific protein U2B″ of T. brucei. Sequence analysis revealed that the trypanosomal orthologue contains only a single RRM, in contrast to the mammalian, two-RRM domain structure, and that the homology is restricted to this single RRM. We show that U2-40K (U2A′) binds very efficiently to U2B″ in the absence of U2 snRNA and increases the binding affinity of U2B″ to U2 snRNA. Furthermore U2-40K (U2A′) contacts the two specific components of the U2 Sm core, Sm16.5K and Sm 15K, forming together a high-affinity, U2-specific binding complex. This establishes a specific function of the U2 Sm core variation in mediating U2-specific protein-protein interactions.     

A Role for SRp54 during Intron Bridging of Small Introns with Pyrimidine Tracts Upstream of the Branch Point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3′ splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3′ splice site. Many small introns, especially those in nonvertebrates, lack a classical 3′ pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5′ splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5′ and 3′ splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3′ pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.Pre-mRNA splicing is a conserved process occurring in a wide variety of eucaryotes with differing exon/intron architectures (reviewed in references 4, 6, 9, 15, 20, and 26). Vertebrates typically have small exons and large introns. Nonmetazoans frequently have the opposite genetic organization, with introns smaller than the minimum permissible for splicing of a vertebrate intron. Drosophila melanogaster possesses a mixture of these two classes of intron sizes (16, 23). In addition, more than half of the small introns in Drosophila are missing a prominent vertebrate splicing signal, the 3′ polypyrimidine tract (23). For these reasons, Drosophila provides a model system in which to study potential mechanistic variations operating during recognition of splicing signals.In the general model of early vertebrate spliceosome complex assembly, U1 snRNP binds to the 5′ splice site and U2 snRNP auxiliary factor (U2AF) binds to the 3′ polypyrimidine tract, thereby facilitating U2 snRNP interaction with the branch point. Various members of the serine/arginine (SR) family of proteins may participate by promoting or stabilizing these interactions (reviewed in references 13, 22, and 31). This family of proteins may also act as exon or intron bridging factors via their SR-mediated interaction with SR domains on the small subunit of U2AF (U2AF³⁵) and the U1 70K protein (32, 33, 38). SF1, originally discovered as an essential splicing factor in reconstitution assays (19), has also been observed to bind to the branch point (7, 8). In yeast, BBP (branch point bridging protein), the ortholog to SF1, functions as an intron bridging factor via interactions with U1 snRNP-associated proteins and the large subunit of U2AF (U2AF⁶⁵) (1, 2). It is assumed that vertebrate SF1 can play a similar role, although the mammalian equivalents to the yeast U1 snRNP proteins that interact with BBP have not yet been identified. Furthermore, the relationship between bridging by SR proteins and that afforded by SF1 is unclear.We have previously examined the cis-acting sequences required for efficient splicing of a constitutively spliced small (59-nucleotide [nt]) intron from the D. melanogaster mle gene that lacks a well-defined pyrimidine tract between the branch point and 3′ splice site (18, 29). Assembly of initial ATP-dependent spliceosomes (complex A) on the mle intron requires both the 5′ and 3′ splice sites, suggesting concerted recognition of the entire intron (29). Instead of a classic pyrimidine tract, the mle intron contains two C-rich tracts located between the 5′ splice site and branch point that are necessary for efficient splicing of this intron (18). In addition to a requirement for maximal splicing efficiency, the pyrimidine stretches are also necessary for binding of U2AF, interaction of factors with the 5′ splice site, and proper assembly of the active spliceosome, suggesting that these sequences affect early assembly events at both ends of this small intron. Interestingly, the upstream C-rich tracts are inhibitory if a classical 3′ pyrimidine tract is introduced between the branch point and 3′ splice site (18). This observation suggests competing pathways of factor binding to this substrate and also raises the possibility of alternative gene-specific modes of association of constitutive factors with introns.Here we demonstrate that both U2AF and an SR protein, SRp54, interact with the C-rich tracts in the mle intron. The central location of the pyrimidine tracts, their importance for maximal splicing, and the ability of human SRp54 to interact with U2AF⁶⁵ instead of U2AF³⁵ (37) suggested that the binding of SRp54 to the tracts could replace SF1 in bridging this intron. Immunoprecipitation studies using an antibody specific for SF1 indicated that SF1 did not contact mle precursor RNA unless a pyrimidine tract was introduced downstream of the branch point. Furthermore, antibodies against either SRp54 or U2AF immunoprecipitated both halves of a precleaved mle splicing substrate, suggesting that these factors either directly or indirectly interact with both the 5′ and 3′ splice sites. We suggest that SRp54 participates in bridging the small mle intron via its ability to bind both the C-rich tracts and the large subunit of U2AF.     

3′ cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications,Deletions, and Substitutions

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3′-noncoding region (3′NCR) of six domains (V, X, I, II-1, II-2, and III in the 5′-to-3′ direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3′NCR in viral replication. The two 3′-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3′NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3′-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3′ cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3′ direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3′NCR of JEV and perhaps in other flaviviruses.Japanese encephalitis virus (JEV), a mosquito-borne flavivirus of the family Flaviviridae, is serologically related to several significant human pathogens, including West Nile virus (WNV), Kunjin virus (KUNV), St. Louis encephalitis virus, and Murray Valley encephalitis virus. It is also phylogenetically close to other clinically important human pathogens, including yellow fever virus (YFV) and dengue virus (DENV) (11, 67). JEV is the leading cause of viral encephalitis in Southeast Asia, including China, Japan, Korea, the Philippines, Thailand, and India, and it has begun to expand throughout the Indonesian archipelago and as far as Australia (21, 43). Despite the fact that JEV is generally asymptomatic, ∼50,000 cases are reported annually, and the disease has a mortality rate of ∼25%, mainly in children and young adults (29, 63). Thus, the geographic expansion and clinical importance of JEV infection have drawn increasing attention from the international public health community (44, 71).Like other flaviviruses, JEV is a spherical enveloped virus (∼50 nm diameter) with a single-stranded positive-sense RNA genome that contains a 5′ cap structure but lacks a 3′ polyadenylated tail. Its genomic RNA of ∼11,000 nucleotides (nt) consists of a single long open reading frame (ORF) with two noncoding regions (NCRs) at the 5′ and 3′ ends (41, 84). The ORF is translated into an ∼3,400-amino acid polyprotein precursor, which is co- or posttranslationally cleaved by a cellular protease(s) or a viral protease complex into 10 mature proteins: (i) three structural proteins, the capsid (C), premembrane (prM; which is further processed into pr and M), and envelope (E) proteins; and (ii) seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, as arranged in the genome (13, 41, 84). The nonstructural proteins, together with cellular factors, form a viral replicase complex that directs the replication of the genomic RNA in the cytoplasm of the host cell in association with perinuclear membranes (40, 74). For the synthesis of the genomic RNA to take place, this replicase complex must specifically recognize viral cis-acting RNA elements, defined by primary sequences or secondary/tertiary structures. These RNA elements are found in various locations within the genome but most frequently are located in the 5′- and 3′NCRs (23, 47). The identification and characterization of these cis-acting RNA elements is critical for understanding the complete cycle of JEV genome replication.The availability of the complete nucleotide sequence of YFV genomic RNA (57) has led to the identification of three major conserved elements in the 5′- and 3′-terminal regions of the genomic RNA that contain the short primary sequences and secondary structures required for flavivirus RNA replication. (i) Both ends of the genomic RNA terminate with the conserved dinucleotides 5′-AG and CU-3′ (9, 10, 32, 45, 57, 72, 73) in all flaviviruses except an insect cell fusing agent virus (12). Mutations substituting another nucleotide for one of these four nucleotides in KUNV or WNV replicon RNA are known to abolish or compromise RNA replication (35, 69). (ii) A 3′ stem-loop structure (3′SL) has been recognized in all flaviviruses within the ∼90-nt 3′-terminal region of the genomic RNA (9, 45, 57). The structural and functional importance of this 3′SL in RNA replication has been demonstrated in several flaviviruses (9, 18, 49, 50, 61, 70, 82, 86). (iii) The presence of short 5′ and 3′ cyclization sequences (5′CYC and 3′CYC, respectively) in all mosquito-borne flaviviruses suggests that flavivirus genomes can cyclize via 5′-3′ long-range base-pairing interaction, since the 3′CYC upstream of the 3′SL is complementary to the 5′CYC in the 5′ coding region of the C protein (30). The role of these CYC motifs in RNA replication has been well characterized via cell-based assays in many mosquito-borne flaviviruses, including KUNV (34), WNV (42), YFV (8, 14), and DENV (2, 22, 49), and in cell-free systems in the case of WNV (51) and DENV (1, 3, 79, 80). Other RNA elements that have recently been shown to be important for RNA replication in DENV and WNV include an additional pair of complementary sequences (designated 5′- and 3′UARs) that participate in genome cyclization (3, 4, 17, 87) and a 5′ stem-loop structure (designated 5′SLA) present within the 5′NCR that promotes RNA synthesis in association with the 3′NCR (22).In all flaviviruses, the 3′NCR of the genomic RNA is relatively long (∼400 to ∼800 nt), with an array of conserved primary sequences and secondary structures. Although significant progress has been made in identifying cis-acting elements within the 3′NCRs that are essential for RNA replication, most of these elements (i.e., the 3′CYC, 3′SL, and CU-3′) are limited to the ∼100-nt 3′-terminal region that is highly conserved in these viruses (see recent reviews in references 23 and 47). However, the functional importance of the remaining 5′-proximal region of the 3′NCR, which differs in sequence between the various serological groups, is poorly understood. In particular, comparative sequence analyses and genetic algorithm-based computer modeling have suggested that in addition to the well-studied ∼100-nt 3′-proximal region, the remaining ∼474-nt 5′-proximal region of the 574-nt JEV 3′NCR also contains several RNA elements that may play critical roles in the viral life cycle (52, 55, 56, 68). To date, however, experimental evidence for the functional importance of these potential RNA elements in JEV genomic RNA replication is lacking.In the present study, we have identified and characterized the 3′ cis-acting RNA elements within the JEV 3′NCR and shown that they play an essential and/or regulatory role in genomic RNA replication. In particular, we have constructed and functionally characterized genome-length JEV mutant cDNAs with a series of 5′-to-3′ or 3′-to-5′ progressive deletions within the 3′NCR. In addition to identifying particular mutations within this region that affect either the competence or efficiency of genomic RNA replication, we found that the serial passaging of these mutants in susceptible BHK-21 cells produced a large number of pseudorevertants bearing a wide variety of spontaneous point mutations and sequence duplications, some of which were capable of restoring the replication competence of the defective mutants or enhancing replication efficiency. In addition, we assessed the replication of these mutants in three different cell types (BHK-21, SH-SY5Y, and C6/36 cells). Collectively, these data offer new insights into the functional importance of 3′ cis-acting RNA elements that regulate the cell type-dependent replication of JEV and perhaps other closely related mosquito-borne flaviviruses. Our findings also provide experimental evidence for the emergence of functional 3′ direct repeat sequences that are duplicated from the coding region and 3′NCR of JEV genomic RNA.