The essential pre-mRNA splicing factor SF2 influences 5′ splice site selection by activating proximal sites

Stage-specific changes in SR splicing factors and alternative splicing in mammary tumorigenesis

“…Neoplasia was accompanied by a dramatic increase in expression of some of the SR family of splicing factors recognized by mAb104, resulting in alteration of the relative abundance of individual SR proteins and an increase in the complexity of expression of this important class of splicing factors. Given the number of genes whose splicing has been observed to be responsive to relative SR protein levels (Ge and Manley, 1990;Krainer et al, 1990b;Ca ceres et al, 1994;Wang and Manley, 1995), this observation suggests that pronounced changes in alternative splicing of a number of pre-mRNAs should accompany mammary tumorigenesis.…”

“…The arginine-serine-rich (SR) proteins ( Figure 1a) constitute a family of essential splicing factors (Krainer et al, 1990a;Ge et al, 1991;Zahler et al, 1992) that recognize both splice sites and exonic splicing enhancers, and in¯uence alternative processing decisions when their relative concentrations are altered in vivo or in vitro (Ge and Manley, 1990;Krainer et al, 1990b;Zahler et al, 1993a;Ca ceres et al, 1994;Wang and Manley, 1995). SR proteins have been observed to in¯uence splicing activity via their binding to both splice sites and special splicing accessory sequences known as enhancers (Zahler et al, 1993b;Fu, 1995;Manley and Tacke, 1996;Valcarcel and Green, 1996).…”

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1

“…The hnRNP A1 variant proteins were assayed for alternative splicing activity using a model b-globin premRNA with a duplicated 59 splice site (Fig+ 4;Reed & Maniatis, 1986;Krainer et al+, 1990)+ Because HeLa cell cytosolic S100 extract contains ;10-fold less hnRNP A1 than nuclear extract (Mayeda et al+, 1993), the proximal 59 splice site is almost exclusively selected in splicing reactions performed in the S100 extract complemented with SF2/ASF (Krainer et al+, 1990)+ Therefore, this complemented extract system was used to obtain maximal switching from the proximal to the distal 59 splice site upon addition of hnRNP A1 wildtype or variant proteins (Fu et al+, 1992;Mayeda & Krainer, 1992;Mayeda et al+, 1994)+ Using the variant recombinant proteins with duplicated or deleted RRMs, we investigated whether the FIGURE 4. Alternative 59 splice-site switching activity of wild-type and variant hnRNP A1 proteins+ Structure of the model b-globin pre-mRNA with duplicated 59 splice sites and the two possible splicing paths are shown schematically at the top+ Splicing reactions contained 6 ml of HeLa cell S100 extract complemented with 0+4 mM purified SF2/ASF plus increasing amounts of the indicated hnRNP A1 wild-type and variant proteins: 0 (Ϫ), 0+3, 0+6, 1+2 mM final protein concentration, respectively+ Positions of the spliced mRNAs generated by selection of proximal or distal 59 splice sites are indicated+ pBR322/Hpa II DNA markers are shown in the first lane with their sizes indicated at left+ individual RRMs of hnRNP A1 have similar properties with respect to promoting distal alternative 59 splicesite selection by antagonizing the activity of SF2/ASF (Fig+ 4)+ Surprisingly, the hnRNP variant comprising a duplicated RRM1, A1-D(RRM1), completely lacked alternative splicing activity, whereas the variant including a duplicated RRM2, A1-D(RRM2), was nearly as active as the wild type+ This striking difference may indicate that RRM2 has a more significant role than RRM1 in distal 59 splice-site selection+ However, an intact two-RRM structure is necessary, because deletion of either RRM, in the A1-⌬RRM1 or A1-⌬RRM2 variants, completely abolished the splice-site switching activity+ Upon swapping the positions of RRM1 and RRM2, in the A1-S(RRM2,1) variant, we observed very weak splicesite switching activity+ We conclude that the presence of at least one copy of RRM2 is essential, and the relative position of RRM1 and RRM2 is important, for efficient activity+ The A1-⌬IRL variant, which has a shortened inter-RRM linker connecting RRM1 to RRM2, was also tested for alternative splicing activity+ This mutant was completely inactive in the alternative splicing assay+ Thus, the IRL is likely to play an important role, either directly by participating in RNA contacts, or indirectly by allowing proper positioning of RRM1 and RRM2+…”

“…The plasmid pSP64-59D16X (Reed & Maniatis, 1986;Krainer et al+, 1990), containing a duplicated 59 splice site, was linearized with BamH I and used as a template for runoff transcription with SP6 RNA polymerase (Mayeda & Krainer, 1998a)+ HeLa cell S100 extract and purified SF2/ASF were prepared as described (Mayeda & Krainer, 1998b;Mayeda et al+, 1993)+ In vitro splicing reactions in 25 ml with the indicated amounts of S100 extract, purified SF2/ASF, the appropriate wild-type or variant recombinant hnRNP A1, and 20 fmol 32 P-labeled pre-mRNA substrate were incubated at 30 8C for 4 h as described (Mayeda & Krainer, 1998a)+ RNA products were analyzed by electrophoresis on a 5+5% polyacrylamide/7 M urea gel followed by autoradiography with an intensifying screen at Ϫ70 8C+…”

“…Following transcription in the cell nucleus, nascent mRNA precursors (pre-mRNAs) rapidly associate with a set of abundant RNA-binding proteins, known as heterogenous nuclear ribonucleoproteins, or hnRNPs+ It is in association with hnRNPs and other less abundant proteins that pre-mRNA molecules undergo processing and subsequent transport from the nucleus to the cytoplasm+ More than 20 proteins, designated hnRNP A to U, have been identified by immunoprecipitation as components of hnRNP complexes in vivo (reviewed in Dreyfuss et al+, 1993)+ The ubiquitous association of hnRNP proteins with pre-mRNA suggests their direct involvement in many different aspects of posttranscriptional nuclear RNA metabolism+ Indeed, hnRNP A1, one of the most abundant core hnRNPs, plays an active role in nuclear-cytoplasmic mRNA transport and in alternative pre-mRNA splicing+ This protein has been shown to shuttle continuously between the nucleus and the cytoplasm (Piñol-Roma & Dreyfuss, 1992)+ Shuttling of hnRNP A1 requires a short C-terminal peptide sequence, termed M9 (Michael et al+, 1995;Siomi & Dreyfuss, 1995;Weighardt et al+, 1995), which interacts specifically with transportin, a component of a receptor-mediated protein import pathway (Pollard et al+, 1996)+ In addition to its role in mRNA transport, hnRNP A1 also plays a role in pre-mRNA alternative splicing+ In transcripts containing alternative 59 splice sites, increasing concentrations of the splicing factor SF2/ASF promote the use of the proximal 59 splice site (Ge & Manley, 1990;Krainer et al+, 1990)+ hnRNP A1 counteracts this activity, thereby promoting use of the distal 59 splice site in vitro and in vivo (Mayeda & Krainer, 1992;Cáceres et al+, 1994;Yang et al+, 1994)+ SF2/ASF is the founding member of a highly conserved set of proteins, termed SR proteins, which are general splicing factors widely distributed among eukaryotes (reviewed in Cáceres & Krainer, 1997)+ hnRNP A1 is the prototype of a structurally related set of proteins, termed the hnRNP A/B family, which also includes hnRNP A1 B , A2, and B1 (reviewed in Dreyfuss et al+, 1993)+ The antagonistic activities in the selection of alternative 59 splice sites are also present to varying degrees in the other members of the SR and hnRNP A/B protein families (Fu et al+, 1992;Mayeda & Krainer, 1992;Mayeda et al+, 1993Mayeda et al+, , 1994Yang et al+, 1994;Shen et al+, 1995)+ Both SF2/ASF and hnRNP A1, as prototypes of the SR and hnRNP A/B proteins, have been well characterized with respect to their RNA-binding properties and their association with pre-mRNA splicing complexes+ However, the mechanisms by which hnRNP A1...…”