Recent structures of the heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) have revealed two unexpected examples of RNA recognition motif (RRM)-like domains with specialized features for protein recognition. These unusual RRMs, called U2AF homology motifs (UHMs), represent a novel class of protein recognition motifs. Defining a set of rules to distinguish traditional RRMs from UHMs is key to identifying novel UHM family members. Here we review the critical sequence features necessary to mediate protein-UHM interactions, and perform comprehensive database searches to identify new members of the UHM family. The resulting implications for the functional and evolutionary relationships among candidate UHM family members are discussed.The processes of RNA splicing, transport, capping, editing, and polyadenylation are heavily dependent on protein factors that recognize the pre-mRNA and assemble the appropriate pre-mRNA processing complexes. Surprisingly, the many different protein factors that guide pre-mRNA modification pathways are composed of a limited number of conserved, modular RNA-binding domains (Burd and Dreyfuss 1994). Of these, the RNA recognition motif (RRM) domain is by far the most abundant type of eukaryotic RNA-binding motif. In addition to associations between protein and RNA, protein-protein interactions are essential to recruit catalytic components to sites of RNA modification and to coordinate pre-mRNA processing with other cellular pathways. Interestingly, traditional protein interaction domains, such as SH2, SH3, and WW motifs, are rarely observed in pre-mRNA processing factors (e.g., see Shatkin and Manley 2000;Zhou et al. 2002), implying that the ability to interact with other proteins may reside in the sequences previously thought to be involved in RNA binding. Consistent with this idea, recent structures of the heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) have revealed two unexpected examples of RRM-like domains with specialized features for protein recognition (Kielkopf et al. 2001;Selenko et al. 2003). In light of this structural information, we call these unusual RRMs U2AF homology motifs (UHMs) to reflect their distinct role in protein recognition. Here, the critical sequence features necessary to mediate protein-UHM interactions are reviewed and formulated in a manner that has permitted a comprehensive database search designed to identify members of the UHM family. The resulting implications for the functional and evolutionary relationships among candidate members of the UHM family are discussed. This review represents a first step toward distinguishing canonical RRMs from UHMs, and thereby contributes toward a major goal of the postgenomic era (Thornton et al. 2000): to convert genomic sequences into testable functional hypotheses. Structural features of RNA recognition by canonical RRMsThe RNA-binding function of the canonical RRM domain has been extensively investigated over the last two decades. The most conserved RRM signature sequence is an eight-resid...

Each of the trypanosome small nuclear ribonucleoproteins (snRNPs) U2, U4͞U6, and U5, as well as the spliced leader (SL) RNP, contains a core of common proteins, which we have previously identified. This core is unusual because it is not recognized by anti-Sm Abs and it associates with an Sm-related sequence in the trypanosome small nuclear RNAs (snRNAs). Using peptide sequences derived from affinity-purified U2 snRNP proteins, we have cloned cDNAs for five common proteins of 8.5, 10, 12.5, 14, and 15 kDa of Trypanosoma brucei and identified them as Sm proteins SmF (8.5 kDa), -E (10 kDa), -D1 (12.5 kDa), -G (14 kDa), and -D2 (15 kDa), respectively. Furthermore, we found the trypanosome SmB (T. brucei) and SmD3 (Trypanosoma cruzi) homologues through database searches, thus completing a set of seven canonical Sm proteins. Sequence comparisons of the trypanosome proteins revealed several deviations in highly conserved positions from the Sm consensus motif. We have identified a network of specific heterodimeric and -trimeric Sm protein interactions in vitro. These results are summarized in a model of the trypanosome Sm core, which argues for a strong conservation of the Sm particle structure. The conservation extends also to the functional level, because at least one trypanosome Sm protein, SmG, was able to specifically complement a corresponding mutation in yeast.T rans splicing in trypanosomes is an essential step in the expression of all mRNAs and results in joining of a short, noncoding miniexon sequence [spliced leader (SL)] to each of the protein-coding sequences that are part of long polycistronic precursors (reviewed in ref. 1). As in the cis-spliceosome, small nuclear RNAs (snRNAs) U2, U4, and U6, in addition to the SL RNA, are essential cofactors for trans splicing (2). In addition, the trypanosomatid U5 snRNA has been identified (3-5). Surprisingly, Schnare and Gray (6) recently discovered also a U1-like small RNA in the trypanosomatid species Crithidia fasciculata and Leishmania tarentolae, which may be required for cis splicing of internal introns such as the one of the poly(A) polymerase gene (7).We had previously established affinity purification procedures that allowed the identification of protein components in the trans-spliceosomal small nuclear ribonucleoproteins (snRNPs) from Trypanosoma brucei. A set of at least five polypeptides of 8.5, 10, 12.5, 14, and 15 kDa, which we have called common proteins, was detected originally in the SL RNP, the U2 snRNP, and the U4͞U6 snRNP (8). Common proteins were localized by immunof luorescence predominantly in the nucleoplasm of trypanosomes (9). They make up a stable core shared between these snRNPs and bind to an snRNA region resembling the Sm sequence of cis-spliceosomal snRNPs (10). Using polyclonal Abs that we generated against a mixture of four of these proteins (8.5, 10, 12.5, and 14 kDa), we showed later that these core proteins are present in the U5 snRNP (4) and the SLA (spliced leader-associated) RNP (11). For the trypanosomal U4 and U5 snRNAs, we ...

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5′‐terminal mini‐exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini‐exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem‐loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini‐exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini‐exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA‐RNA interactions in the trans spliceosome.

Processing of primary transcripts in trypanosomes requires trans splicing and polyadenylation, and at least for the poly(A) polymerase gene, also internal cis splicing. The trypanosome U1 snRNA, which is most likely a cis-splicing specific component, is unusually short and has a relatively simple secondary structure. Here, we report the identification of three specific protein components of the Trypanosoma brucei U1 snRNP, based on mass spectrometry and confirmed by in vivo epitope tagging and in vitro RNA binding. Both T.brucei U1-70K and U1C are only distantly related to known counterparts from other eukaryotes. The T.brucei U1-70K protein represents a minimal version of 70K, recognizing the first loop sequence of U1 snRNA with the same specificity as the mammalian protein. The trypanosome U1C-like protein interacts with 70K directly and binds the 5′ terminal sequence of U1 snRNA. Surprisingly, instead of U1A we have identified a novel U1 snRNP-specific protein, TbU1-24K. U1-24K lacks a known RNA-binding motif and integrates in the U1 snRNP via interaction with U1-70K. These data result in a model of the trypanosome U1 snRNP, which deviates substantially from our classical view of the U1 particle and may reflect the special requirements for splicing of a small set of cis-introns in trypanosomes.

Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNPspecific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro proteinprotein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.

Ullu, 1990) and occur in the form of snRNPs in the cell S.Lücke, T.Klöckner and Z.Palfi contributed equally to this work (Michaeli et al., 1990;Cross et al., 1991;Günzl et al., 1992). The SL RNP is an additional, trans-splicing specific In trypanosomes all mRNAs are generated through factor and has been proposed as a U1 snRNP equivalent trans mRNA splicing, requiring the functions of the (Bruzik et al., 1988; discussed in Steitz, 1992); although small nuclear RNAs U2, U4 and U6. In the absence of it contributes its miniexon during the trans-splicing reacconventional cis mRNA splicing, the structure and tion, it also resembles an snRNP on the basis of its protein function of a U5-analogous snRNP in trypanosomes composition . Using affinity purification has remained an open question. In cis splicing, a U5of Trypanosoma brucei snRNPs, five common lowsnRNP-specific protein component called PRP8 in yeast molecular-weight polypeptides have been identified that and p220 in man is a highly conserved, essential splicing are shared by the U2, U4/U6 and SL RNPs; in addition, factor involved in splice-site recognition and selection.this analysis revealed several snRNP-specific protein comWe have cloned and sequenced a genomic region ponents . In contrast to the yeast and from Trypanosoma brucei, that contains a PRP8/p220-mammalian cis-splicing systems, only one snRNP protein homologous gene (p277) coding for a 277 kDa protein.gene has been cloned so far from the trypanosome transUsing an antibody against a C-terminal region of splicing system, coding for a U2-specific 40 kDa protein the trypanosomal p277 protein, a small RNA of~65 with homology to the cis-spliceosomal U2 AЈ protein nucleotides could be specifically co-immunoprecipit- . ated that appears to be identical with a U5 RNA (SLA2 An important open question has been whether there is RNA) recently identified by Dungan et al. (1996). Based a trans-spliceosomal homologue of the U5 RNA. U5 RNA on sedimentation, immunoprecipitation and Western is the least conserved among the spliceosomal RNAs, and blot analyses we conclude that this RNA is part of a phylogenetic comparisons showed that essentially only stable ribonucleoprotein (RNP) complex and associated the 11-nucleotide 5Ј loop sequence is conserved (Guthrie not only with the p277 protein, but also with the and Patterson, 1988;Frank et al., 1994). The 5Ј loop of common proteins present in the other trans-spliceo-U5 RNA is functionally important in cis splicing, in somal snRNPs. Together these results demonstrate particular during 5Ј and 3Ј splice-site recognition and that a U5-analogous RNP exists in trypanosomes and selection; this has been established in yeast using in vivo suggest that basic functions of the U5 snRNP are mutational studies Norman, 1991, 1992) conserved between cis and trans splicing.and also in the mammalian splicing system (Wyatt et al., Keywords: snRNA/snRNP/trans splicing/trypanosomes/ 1992; Cortes et al., 1993;Sontheimer and Steitz, 1993

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