U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Abstract

Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (A_AG $ GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/US/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.[Key Words: 5' splice site recognition; spliceosome assembly~ snRNPs] Received May 13, 1994~ revised version accepted July 1, 1994.Removal of introns from nuclear precursor messenger RNA {pre-mRNA} is mediated by a large multicomponent complex called the spliceosome which is composed of U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs} and numerous proteins (for review, see Green 1991; Guthrie 1991; Moore et al. 1993).In mammals, spliceosome assembly depends on a consensus sequence at the 5' splice site (5'SS}, a branch site with an adjacent polypyrimidine tract, and a 3'SS consensus sequence. Initially, U 1 snRNP binds via base pairing with the 5'SS and commits the pre-mRNA to the splicing pathway. Subsequently, U2 snRNP binds at the branch site to form splicing complex A, which is converted into splicing complex B upon association of U4/ U5/U6 triple snRNP. Within U4/U5/U6 snRNP, U4, and U6 snRNAs are extensively base paired. Prior to {or concomitant with} the first step of splicing, this U4-U6 base pairing is disrupted and U4 snRNP is released. Because the pairing interaction between U6 and U4 snRNAs has been proposed to negatively regulate U6 and is mutually exclusive with the pairing interaction between U6 and U2 snRNAs, this structural rearrangement could represent the catalytic activation of the spliceosome.

Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

“…The multiple recognition events of the 59 splice site make it difficult to explore the contribution from individual factors to the sequence specificity+ To investigate the contribution from other factors than the 59 end of the U1 snRNA, we repeated the selection experiment in the presence of 59 truncated U1 snRNA that could not engage in base pairing with the 59 splice site+ Surprisingly, the consensus 59 splice site sequences after one and three rounds of selection were highly similar to the motifs obtained in the presence of intact U1 snRNA+ It is unlikely that the trace of intact U1 snRNA in the ⌬59end U1-NE contributes significantly to the splicing, as the complex A formed in this extract contained less than 5% intact U1 snRNA+ Moreover, the observed complex A most likely constitutes a functional prespliceosome complex because it was converted into a fully assembled spliceosome (complex B) at the same rate as the appearance of splicing products (Fig+ 7B, right panel)+ Notably, splicing of the 3 ϩ 8 clone in ⌬59end U1-NE reached a level of splicing efficiency after 90 min that was almost indistinguishable from splicing in normal extract+ This suggests that the 59 end of the U1 snRNA is dispensable for splicing and that other factors recognize the entire 59 splice consensus sequence+ Because we, in this study, select for turnover of splicing substrate, the consensus 59 splice site sequence may be shaped both by functional interactions with transacting snRNA and protein factors and as a cisacting sequence with importance for the catalytic step+ One candidate protein factor that potentially could contribute to the sequence specificity is the U1 snRNP specific protein U1C+ Complementation studies with purified U1 snRNP particles lacking subsets of U1-specific proteins show that U1C, but not U1-70K and U1A, is important for formation of early spliceosome complexes in mammalian systems (Heinrichs et al+, 1990;Jamison et al+, 1995;+ Moreover, U1C can be crosslinked to the 59 splice site both in the mammalian (Rossi et al+, 1996) and yeast systems (Zhang & Rosbash, 1999)+ In yeast, the yU1-70K, ySmD1, ySmD3, ySmB, Nam8, and Snu56 proteins also crosslink to the 59 splice site (Zhang & Rosbash, 1999) and the Sm proteins have been shown to stabilize U1 binding (Zhang et al+, 2001)+ Another candidate factor is the U5 snRNP-specific protein Prp8 based on numerous reports of Prp8 crosslinks to the 59 splice site (Wyatt et al+, 1992;Teigelkamp et al+, 1995;Reyes et al+, 1999;Siatecka et al+, 1999;Collins & Guthrie, 1999;Maroney et al+, 2000)+ The binding of Prp8 to the 59 splice site is probably responsible for the recruitment of U4/U6+U5 tri-snRNP to the spliceosome (Konforti & Konarska, 1994) and more recent data suggests that this represents an important ATP-dependent step in early spliceosome assembly (Maroney et al+, 2000)+ During multiple rounds of selection,...…”

The 100-kDa U5 snRNP protein (hPrp28p) contacts the 5′ splice site through its ATPase site

“…In HeLa cell extracts, the 59SS:hPrp28p crosslink forms at the stage of complex B assembly and remains detectable throughout the reaction, suggesting that the relative positions of hPrp28p and the 59SS remain mostly unchanged during splicing+ This is in sharp contrast to the crosslinking profile observed for the 59SS:hPrp8p interaction at the 59 splice site junction (Reyes et al+, 1999), which was characterized only by a transient signal at the initial stages of complex B formation+ The site of contact between the 59SS and hPrp28p is located Crosslinking reactions were conducted with extracts containing intact U1 snRNA (lanes 1 and 2) or with RNase H-treated extracts in the presence of DNA oligonucleotides complementary to the 59 end of U1 snRNA, in the absence (lanes 1 and 3) or in the presence of ATP (lanes 2 and 4)+ The resulting 59SS:hPrp28p and 59SS:PSF crosslinks were analyzed in a 13% SDS gel (C), whereas the 59SS:U1 and 59SS:U6 snRNA crosslinks were resolved in a 10% polyacrylamide/8 M urea gel (D)+ Positions of the 59SS:hPrp28p, 59SS:PSF, 59SS:U1, 59SS:U6 snRNA crosslinks, and the molecular weight markers are indicated+ only a few nucleotides away from the 59SS:hPrp8p interaction at the conserved GU dinucleotide (Reyes et al+, 1996), suggesting that hPrp28p and hPrp8p must be positioned in a close proximity of each other within the U4/U5/U6 snRNP (Fig+ 7)+ Indeed, a genetic interaction has been demonstrated between yeast Prp8p and Prp28p (Strauss & Guthrie, 1991)+ The 59SS: hPrp28p crosslink is detectable both in cis-and transsplicing systems+ Because the main difference between these in vitro systems concerns the initial 59SS:U1 snRNA pairing, which is required for cis-splicing but bypassed in trans-splicing reactions, we conclude that the 59SS:hPrp28p interaction does not strictly depend on this step in spliceosome assembly+ Although the integrity of U6 snRNA is essential for the 59SS:hPrp28p crosslink in both trans-and cis-splicing reactions, intact U2 snRNA is required only under cis-splicing conditions (Fig+ 3), suggesting that the 59SS:hPrp28p contact occurs already within U4/U5/U6 snRNP and does not strictly require U2 snRNP+ Similarly, in transsplicing reactions, formation of the 59SS:U6 snRNA duplex requires intact U6, but not U2 snRNA (Konforti & Konarska, 1994; Fig+ 3)+ Positions within the 59SS RNA that support crosslinking to hPrp28p (peak at positions ϩ7) fall within the region involved in the 59SS:U6 snRNA pairing interaction (positions 40-42 in hU6 snRNA; Wassarman & Steitz, 1992), suggesting that hPrp28p contacts not the 59SS alone, but rather the 59SS:U6 FIGURE 6. Mapping of the site of the 59SS RNA crosslink within hPrp28p+ A: A 300-mL crosslinking reaction using the 32 P-labeled BP-derivatized 59SS RNA was spun through a 900 mL cushion of buffer D and the pellet was treated with RNase T2+ Aliquots (5 mL) from the reaction, before (lane 1) and after RNase T2 treatment (lane 2), were resolved in a 13% polyacrylamide SDS gel to control for RNase T2 digestion+ B: The 59SS:hPrp28p crosslink was gel purified and digested with Glu-C (lane 1) protease, or treated with cyanogen bromide (CnBr) either at 25 8C (lane 2) or at 37 8C (lane 5)+ The acid treatment was carried out in the presence of 13% acetic acid (lane 3) or 13% acetic acid, 6 M guanidinium chloride (lane 4)+ Digestion products were resolved in a 22+5% SDS gel and visualized by autoradiography+ C: Enzymatic digestions of the 59SS:hPrp28p crosslink with Lys-C (lane 1), Glu-C (lane 2), and Arg-C (lane 3) proteases+ Digestion products were resolved in a 16% Tris-Tricine gel+ Positions of the molecular ...…”

“…Proposed scheme for interactions between the 59SS and the spliceosomal components leading to the stable 59SS:U4/U5/U6 snRNP association+ Under conditions when the 59SS is prevented from base-pairing with U1 snRNA, and in the absence of ATP, the 59SS interacts with PSF, most likely in a semistable association with U4/U5/U6 snRNP+ Upon hPrp28p-mediated ATP binding and hydrolysis, the 59SS is translocated into the complex, resulting in a basepairing interaction with U6 snRNA+ At this stage the 59SS junction is known to interact with hPrp8p+ The relative positions of the components are arbitrary+ See text for further details+ 59SS:U6 snRNA:hPrp28p interaction+ Because hPrp28p belongs to the DEAD-box family of putative helicases, the 59SS:hPrp28p crosslink requires the presence of NTP, and maps to the conserved motif III expected to lie in proximity of the RNA pulled into the protein in the NTP-dependent fashion; the simplest interpretation is that the 59SS:hPrp28p contact results directly from hPrp28p-mediated NTP and RNA binding (Fig+ 7)+ It is tempting to speculate that this reaction constitutes the ATP-dependent step observed at the stage of the 59SS:U4/U5/U6 snRNP interaction (Konforti & Konarska, 1994;Maroney et al+, 2000)+ At the moment, no other NTP-dependent steps are known to operate at this stage of splicing, although their existence cannot be excluded+ It is possible that in addition to hPrp28p, other DExD/H-box spliceosomal proteins are involved+ One possible candidate is the U5-200-kDa protein, a human ortholog of S. cerevisiae Brr2p/Snu246p/Prp44p, that exhibits an ATPase/RNA helicase activity in vitro (Lauber et al+, 1996;Kim & Rossi, 1999)+ The ATPase/ RNA helicase activities required for complex A formation are unlikely to be involved here, as U2 snRNP is not required for association of the 59SS with U4/U5/U6 snRNP and the 59SS:hPrp28p interaction+ Implications for the positioning of the 59 splice site within the spliceosome…”

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

“…It is also known that U2 snRNP can bind independently of other snRNPs to the BS (for examples see 37,38) and to the 5′ SS (34), and that the U4/U5/U6 snRNP can bind to the 5′ SS independently of U2 snRNP (23,71). Yet it is still unclear whether U2 must bind to the BS before U2/U6 helix II can form.…”