Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5′ Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking

Abstract

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5 splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5 splice site. Moreover, although p65 cross-linking requires only a 5 splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.Splicing of pre-mRNA occurs within the multicomponent spliceosome complex via two successive chemical steps (reviewed in references 33, 41, 47, and 63). Introns are excised and adjacent exons are spliced together, and this must be achieved with great accuracy, even though the introns may be many thousands of bases in length. Sequence elements at the splice sites themselves, as well as activating enhancer elements, provide the signals within the pre-mRNA that give rise to this specificity. The requirement for accuracy in determination of the precise splice junctions is reflected in a number of steps in which the primary consensus elements are recognized sequentially by separate protein or small nuclear RNA (snRNA) splicing factors. Work with both yeast and HeLa nuclear extracts has led to a model in which the spliceosome is assembled by sequential addition of small nuclear ribonucleoprotein particles (snRNPs) and other splicing factors and by substantial conformational rearrangements before the actual catalytic steps (reviewed in reference 58). A number of the steps leading to the formation of a catalytically competent spliceosome require ATP hydrolysis, either for protein phosphorylation by kinases or for the action of RNA helicases. Initial recognition of splice sites involves binding of U1 snRNP to the 5Ј splice site, mediated by base pairing between the 5Ј splice site consensus sequence and the 5Ј end of U1 snRNA (62,64,84). At the 3Ј end of the intron, the heterodimeric protein U2AF binds to the polypyrimidine tract via the RNA binding domains of the 65-kDa subunit (...

p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

“…To characterize the identity of p65, a number of immunoprecipitation experiments with antibodies against the known splicing factors with similar molecular mass have been carried out. Antibodies against U1-70K, U2AF65, p69, and SF1 were not able to precipitate p65 (25). Another candidate is p68 RNA helicase, which has recently been shown to be a potential spliceosomeassociated protein (31).…”

“…Immunoprecipitation experiments were performed as described in previous studies (24,25). Briefly, MBmediated cross-linking was carried out with RNA GCϩDX/XhoI in 30% HeLa nuclear extracts.…”

“…Due to the unique specificity of the methylene blue (MB)-mediated cross-linking method (26), a 65-kDa protein (p65) was detected interacting with the U1-5Јss duplex during the splicing process (25). p65 interacts with the U1-5Јss duplex at the exon and intron junction, and the intact U1 snRNP is required for the interaction.…”

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

“…Interactions between the 59 splice site of the pre-mRNA and U1, U5, or U6 snRNAs that are supported by experiments+ U1 snRNA is believed to form a transient interaction with the 59 splice site prior to splicing and is replaced by U6 and U5 snRNAs that interact with the same sequences at the time splicing occurs+ Functional selection of 59 splice sites 167 are unknown (Liu et al+, 1998;Sha et al+, 1998;Ismaili et al+, 2001)+ A picture emerges where a given 59 splice site region is recognized through a complex network of interactions that changes during the course of splicing+ We have previously used an iterative functional selection approach to study the recognition of the branch point and 39 splice site (Lund et al+, 2000)+ Here we extend these studies by developing an in vitro strategy for iterative selection of functional 59 splice site sequences from a pool of pre-mRNAs containing randomized inserts+ We provide strong evidence for the hypothesis that factors other than the 59 end of the U1 snRNA determine the shape of the 59 splice site+ Moreover, based on splicing studies of individual pre-mRNAs with variable complementarity to the U1 snRNA, we conclude that stabilizing U1 snRNA binding to the 59 splice site in the natural range increases the competitive strength of a splice site, but concurrently inhibits the assembly of the full spliceosome+…”

Characterization of U4 and U6 interactions with the 5′ splice site using a S. cerevisiae in vitro trans-splicing system

“…These events are thought to be initiated when, in the absence of ATP, the U1-snRNP particle associates with the 5Ј splice site via base-pairing with the 5Ј end of the U1 snRNA (Mount 1982;Zhuang and Weiner 1986;Seraphin et al 1988;Siliciano and Guthrie 1988) and association of U1-snRNP proteins with sequences around the 5Ј splice site (Liu et al 1998;Zhang and Rosbash 1999). In what has been thought to be the first energy-requiring step, U2 snRNA base-pairs with the pre-mRNA branch-point sequence (Cheng and Abelson 1987;Parker et al 1987;Krämer 1988;Wu and Manley 1989;Zhuang and Weiner 1989;Pruzan et al 1990;Michaud and Reed 1991;Liao et al 1992).…”