Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP

Branchpoint selection in the splicing of U12-dependent introns in vitro

“…U12-and U6atac-specific crosslinks form with the SME, XTF, and XRP substrates+ Splicing reactions containing 29-O-methyl oligonucleotides complementary to U12 (U12a, or a above the lane) or U2 (U2b, or b above the lane) were exposed to 365 nm light (indicated by ϩ above the lane) in the presence of psoralen+ Purified RNA was resolved on a 5% denaturing gel, transferred to nylon membrane, and the resulting northern blot probed for U12 snRNA+ A cross-reacting band (*) detected even in the absence of UV irradiation either ran below or comigrated with the lower U12•U6 atac crosslink (helix Ib), depending on the experiment+ Lane 1 contains a molecular weight marker, a 32 P-labeled pBR322 MspI digest+ Lane 2 is a control reaction without any added substrate RNA+ (Fig+ 1, lanes 7 and 8) forms a crosslink with U12 that has a faster mobility, as predicted from its smaller size+ Again, this crosslink and the other two crosslinks diagnostic of the active site helices Ia and Ib between U12 and U6 atac appear only in the absence of the U12-blocking oligonucleotide (Fig+ 1, compare lanes 8 and 9)+ XRP (Fig+ 1, lanes 12 and 13) and XTF (Fig+ 1, lanes 17 and 18) form similar U12•BPS and U12•U6 atac crosslinks in a U12-dependent fashion (Fig+ 1, lanes 14 and 19), although the lower U12•U6 atac crosslink is partially obscured by a cross-reacting band in this particular U12-probed northern blot (Fig+ 1, lanes 10-19)+ In earlier experiments, we found that constructs containing the full-length introns of XRP and XTF similarly formed U12•BPS and U12•U6 atac crosslinks (data not shown), consistent with excision via the U12-dependent pathway+ The truncated XRP and XTF splicing substrates were used in this and later experiments because of their enhanced splicing activity and because their lariat intermediates were readily resolved in 10% gels, thus allowing branch site mapping by primer extension (see below)+ In other respects, the three introns behave like other U12-dependent introns studied+ Stimulation of splicing by the presence of flanking introns, as described for the sodium channel intron (Wu & Krainer, 1998, was confirmed by adding an upstream polypyrimidine tract or a downstream U1 binding site; in each case, a two-to fivefold increase in spliceosome assembly as measured by U12-dependent crosslinks was observed (data not shown)+ Chimeric substrates created from P120 and XRP sequences to test the role of a purinerich sequence in the 59 exon of P120 exhibited moderately enhanced spliceosome assembly+ Exonic enhancers have been demonstrated to modulate U12-type splicing (Dietrich et al+, 2001b;Hastings & Krainer, 2001), and this putative exonic splicing enhancer fits the GAR repeat consensus for SR protein binding (Lavigueur et al+, 1993;Sun et al+, 1993;Watakabe et al+, 1993;Ramchatesingh et al+, 1995…”

Determinants of Plant U12-Dependent Intron Splicing Efficiency

“…The best described of these factors are the SR proteins, a large family of polypeptides with one or more RNA binding domains and a variable length domain (the RS domain) containing multiple copies of Arg-Ser dipeptides (Fu, 1995;Valcá rcel and Green, 1996;Wang and Manley, 1997;Tacke and Manley, 1999;Black, 2003). Although purine-rich enhancers were identified in many exons flanking U2-dependent introns, it already has been demonstrated, both in vitro and in vivo, that ESEs of this type are also functional in the U12 pre-mRNA splicing pathway (Wu and Krainer, 1998;Graveley, 2000;Dietrich et al, 2001b;Hastings and Krainer, 2001). Many SR proteins have been identified in plants (Lazar et al, 1995;Lopato et al, 1996aLopato et al, , 1996bLopato et al, , 1999aLopato et al, , 1999bLopato et al, , 2002, but to date, only one purine-rich exonic element, which promotes 59 splice site selection, has been described (McCullough and Schuler, 1997).…”

Functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway

“…To identify the splicing components that are missing from S100 extract and to establish a system for testing SR protein requirements in AT-AC splicing, we fractionated nuclear extract using selective precipitation by ammonium sulfate+ Three nuclear extract fractions were generated by precipitation at 20% ammonium sulfate saturation, followed by precipitation at 60% ammonium sulfate (20-60%AS) and precipitation at 90% saturation (60-90%AS)+ Splicing reactions containing both the 20-60%AS and 60-90%AS fractions reconstituted b-globin and SCN4A splicing (not shown)+ SCN4A splicing was not observed with either fraction alone (Fig+ 1, lane 3 and not shown) or when either fraction was added to S100 extract (Fig+ 1, lane 4 and not shown)+ Because the bulk of SR proteins is found in the 60-90%AS fraction (Zahler, 1999 and not shown), the lack of splicing in S100 extract with the 20-60%AS fraction may be due to the absence of SR proteins in this fraction+ Indeed, the SCN4A AT-AC intron was spliced when SR proteins were included with S100 extract and 20-60%AS (Fig+ 1, lanes 6 and 7)+ This splicing reaction was as efficient as SCN4AS splicing in nuclear extract (Fig+ 1, lane 1) and clearly demonstrates the requirement for SR proteins in SCN4A AT-AC intron splicing+ SR proteins mediate splicing in basal and enhancer-dependent AT-AC splicing SCN4A AT-AC intron splicing is stimulated by a downstream 59ss or an exonic splicing enhancer (Wu & Krainer, 1996)+ To determine if SR proteins mediate splicing of the basal SCN4A substrate and support stimulation of splicing by enhancer elements, splicing of three different SCN4A pre-mRNA substrates was compared+ SCN4AS is described above, SCN4Aϩ59ss is identical to SCN4AS but has a conventional downstream 59ss following exon 3 (Wu & Krainer, 1997) and SCN4AϩEnh contains a synthetic purine-rich exonic splicing enhancer at the end of exon 3, but no downstream 59ss (Wu & Krainer, 1998)+ All three of the AT-AC pre-mRNA substrates were spliced in nuclear extract (Fig+ 1, lanes 1, 8, and 15), though less accumulation of mRNA products was observed with SCN4AS relative to SCN4Aϩ59ss and SCN4AϩEnh+ This result is consistent with the previous study (Wu & Krainer, 1998) and confirms the activity of the downstream 59ss and enhancer sequence in AT-AC intron splicing+ The level of splicing stimulation by these elements was approximately twofold relative to the basal substrate+ This stimulation is lower than that reported previously (Wu & Krainer, 1996 because the experiments presented here were performed under splicing conditions optimized in favor of the basal splicing reaction, which has a narrower optimal concentration of magnesium than the other substrates+ To determine the requirement for SR proteins in splicing of these RNA substrates, the reconstituted system described above was used+ In addition to the basal SCN4A substrate, SCN4A splicing also was observed in the presence of a downstream 59ss or enhancer sequence upon addition of SR proteins and the 20-60%AS fraction to S100 extract (Fig+ 1, lanes 6-7, 13-14, and 20-21)+ The splicing efficiencies of SCN4Aϩ 59ss and SCN4AϩEnh in the reconstituted system nearly equaled those in nuclear extract+ Splicing of SCN4Aϩ59ss and SCN4AϩEnh pre-mRNAs was more efficiently complemented by SR proteins than splicing of the basal SCN4A substrate, as indicated by the greater accumulation of mRNA products (cf+ Fig+ 1, lanes 6, 13, and 20)+ These results suggest that SR proteins not only function in basal AT-AC spli...…”