sans fille

Protein Interactions

A complete set of seven U1-related sequences have been cloned and characterized from Drosophila melanogaster. These sequences, coding for U1 snRNA's, the RNA component of U1A, are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D that encodes the prototype U1 sequence (U1a), one U1 gene at 82E that encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes comprise another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C that encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. The U1b RNA is expressed in Drosphila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. The expression throughout Drosophila development of the two U1 variants has been observed relative to the prototype sequence. The U1c variant is undetectable, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse (Lo, 1990).

Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position -40, relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity of Drosophila snRNA genes is intrinsically encoded in the PSE sequence itself. The differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs has been investigated. By using a site specific protein-DNA photo-cross-linking assay, three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa have been identified that are in close proximity to the sequence element. Two additional putative polypeptides of 230 and 52 kDa have been identified that may be integral to the complex. The 95-kDa subunit cross-links at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-links only to the 3' half of the PSE. The same polypeptides cross-link to both the U1 and U6 PSE sequences. However, there are significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding is to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters (Wang, 1998).

Sans fille (SNF), a Drosophila homolog of mammalian U1A and U2B" snRNP proteins, is an integral component of the machinery required for splice site recognition in all pre-mRNAs. Mutations in snf disrupt the establishment of Sex lethal mRNA female-specific splicing pattern in both the germ line and soma. Because snf is required to establish the female-specific splice site choice, snf is likely to function in conjunction with SXL to bias the general splice machinery against the recognition of the male 5' splice site. SNF cooperates with SXL to block utilization of the male-specific exon of the SXL pre-mRNA, suggesting a model in which the SXL protein blocks spliceosome assembly by forming a non-productive snRNA/SXL complex. This suggests that snRNPs, like transcription factors, can have antagonistic roles in controlling gene expression (Salz, 1994 and Salz, 1996).

SXL and SNF proteins can interact directly in vitro, and these proteins are part of an RNase-sensitive complex in vivo that can be immunoprecipitated with anti-SXL antibody. The SNF protein associated with SXL protein is in a large, rapidly sedimenting complex. These complexes contain additional small nuclear ribonucleoprotein particle protein and the U1 and U2 small nuclear RNAs. Sxl transcripts can also be immunoprecipitated by anti-SXL antibodies. A model is presented for SXL mRNA splicing regulation. SXL protein binds to intron sequences far from the male exon (exon 3) and blocks the utilization of male exon splice sites. SXL proteins would bind to the poly U tracts in the introns upstream and downstream of the SXL male exon. SXL proteins in the intron would then make contact with the U1 and U2 snRNPs associated with the male exon splice junctions, presumably via protein-protein interactions with SNF. These contacts would prevent the snRNPs at the male exon splice junctions from participating in subsequent splicing steps (Deshpande, 1996).

Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. The splicing factors SC35 and SF2/ASF, both functioning as SR proteins, specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing (Wu, 1993).

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. A splicing 'coactivator', SRm160/300, has been identified that contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independent of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound 'activators' and the snRNP machinery of the spliceosome (Eldridge, 1999).

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins that stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. This model was tested by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the Drosophila doublesex gene. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) binds equally to the pre-mRNA in either the presence or absence of the ESE. Time course experiments reveal differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA: U2AF-65 kDa binds prior to most SR proteins and hTra2b and its level of binding does not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA is, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract (Li, 1999).

The factor requirements for binding of U2AF-65 kDa to the dsx substrate were investigated. U1 snRNP is required for the binding of U2 snRNP to the dsx(GAA)₆ pre-mRNA used in the present study. U1 snRNP can promote the cross-linking of U2AF-65 kDa to an upstream polypyrimidine tract across an exon and also to the polypyrimidine tract of a constitutively spliced pre-mRNA containing a single intron. Would U1 snRNP also be required for the binding of U2AF-65 kDa, within the context of cross-intron interactions during ESE-dependent splicing on the biotinylated dsx(GAA)₆ pre-mRNA? Splicing complexes assembled on this substrate were affinity selected from splicing reactions depleted of individual snRNPs and then immunoblotted with the anti-U2AF-65 kDa antibody. Depletion of U1 snRNP results in a significant reduction in the level of U2AF-65 kDa binding to the dsx(GAA)₆ pre-mRNA compared with its level of binding in a 'mock'-depleted extract. This reduction is not due to a nonspecific loss since depletion of U2 snRNP does not reduce the level of U2AF-65 kDa binding, and mixing equal amounts of the U1 and U2 snRNP-depleted extracts restores binding to the level observed in the mock-depleted reaction. These results indicate that U1 snRNP functions in stabilizing the binding of U2AF-65 kDa to the dsx(GAA)₆ pre-mRNA (Li, 1999).

It is proposed that U1 snRNP promotes two distinct sets of interactions during ESE-dependent splicing. One set involves ESE-independent interactions that are required for the binding of U2AF-65 kDa to the polypyrimidine tract, which then promotes partial binding of U2 snRNP to the branch site. This set of interactions likely involves cross-intron interactions mediated by the branch site-binding factor SF1/mBBP, which interacts with U2AF-65 kDa and is also required for the stable binding of U2 snRNP to the branch site. The other set of interactions promoted by U1 snRNP simultaneously requires the ESE and functions to further stabilize the binding of U2 snRNP to the branch site; this set of interactions also promotes the association of SRm160/300 with the pre-mRNA. This set of interactions does not influence the binding of U2AF to the pre-mRNA. Although depletion of SRm160/300 or U2 snRNP weakens but does not prevent the association of the other component with the dsx(GAA)6 pre-mRNA, these two components interact specifically. Thus, instead of promoting splicing complex formation through interactions mediated by the U2AF heterodimer, one or more ESE-associated components, including the SRm160/300 splicing coactivator, may promote splicing by interacting directly with the snRNP machinery of the spliceosome (Li, 1999).

Transcriptional regulation of U1 small nuclear RNA gene, encoding the RNA component of U1 snRNP

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, and located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduces transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increases the efficiency of the U1 promoter and simultaneously relieves the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter has no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter (Zamrod, 1993)

Most of the major spliceosomal small nuclear RNAs (snRNAs) (i.e. U1, U2, U4 and U5) are synthesized by RNA polymerase II (pol II). In Drosophila melanogaster, the 5'-flanking DNA of these genes contains two conserved elements: the proximal sequence element A (PSEA) and the proximal sequence element B (PSEB). The PSEA is essential for transcription and is recognized by DmSNAPc, a multi-subunit protein complex. Previous site-specific protein-DNA photo-cross-linking assays have demonstrated that one of the subunits of DmSNAPc, DmSNAP43, remains in close contact with the DNA for 20 bp beyond the 3' end of the PSEA, a region that contains the PSEB. Mutation of the PSEB does not abolish the cross-linking of DmSNAP43 to the PSEB. Thus the U1 PSEA alone is capable of bringing DmSNAP43 into close contact with this downstream DNA. However, mutation of the PSEB perturbs the cross-linking pattern. In concordance with these findings, PSEB mutations result in a 2- to 4-fold reduction in U1 promoter activity when assayed by transient transfection (Lai, 2005).

Conserved elements analogous to the PSEB have not been identified in the Pol II-transcribed snRNA genes of other metazoans in which functional studies have been carried out. However, snRNA genes of other insects contain conserved nucleotides in this location. It is possible that fruit flies (and other insects) have taken advantage of utilizing the specificity of the PSEB to modulate the strength of snRNA promoters over an evolutionary time scale. For example, three variant U5 genes, which are probably expressed at low levels, have PSEBs that are among the most divergent from the consensus PSEB (Lai, 2005).

Interestingly, substitution mutations in the -33 to -20 region of a human U2 gene have been found to have minor effects on the transcription start site. It is therefore possible that the D. melanogaster PSEB, which is located within this region, may play a role in helping to establish the correct start site. Transcription of the D. melanogaster U1 gene has been shown to require the TBP. Due to the location of the PSEB (-25 to -32) and its 8 bp length, it seems possible that the PSEB may be a site of DNA interaction with TBP. The PSEB may represent a 'compromise' sequence that allows it to be co-occupied simultaneously both by DmSNAP43 and TBP (Lai, 2005).

To see if this might be possible, TBP bound to DNA was modeled as if the PSEB were a TATA-box. Then, taking into consideration that the PSEA is separated from the PSEB by exactly 8 bp, the sites were identified where DmSNAP43 would cross-link with the DNA. The modeling illustrates that the phosphates that cross-link to DmSNAP43 are not occluded by TBP, and further suggests that DmSNAP43 could interact with the DNA both 'behind' and 'beneath' TBP. Further experiments will be required to examine the validity of this working model (Lai, 2005).

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