Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

Abstract

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of Ul small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of Ul snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slowmigrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact Ul and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. Ul snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by Ul-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H deavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions. Understanding the mechanism of premessenger splicing will require delineation of the pathway of assembly and disassembly of the spliceosome during the splicing reaction. Single-intron substrates of 400 to 500 nucleotides rapidly assemble into large ribonucleoprotein particle (RNP) complexes when added to in vitro splicing extracts (2, 6, 8, 12-15, 17-19, 20a, 22, 23, 26-29, 31). Fractionation of splicing reactions by sucrose gradient sedimentation reveals several different complexes (2,6,8,12,13,17,26,29; Kramer, in press). The largest complex of 50S contains precursor RNA and the reaction intermediates, exon 1 and lariat-exon 2. This complex contains small nuclear RNPs (snRNPs), including Ul snRNPs (2,8,(12)(13)(14), and undergoes rapid splicing to products without a detectable lag when added back to extract (13). Furthermore, it does not form in the absence of ATP or with substrates that lack splicing consensus sequences (2,8,12,13). On the basis of these characteristics, this complex can be considered as an active spliceosome. Smaller assemblies of 20S and 35S have also been noted which rapidly form with precursor RNA and which may be kinetic ...

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

“…In supportive experiments, we also microinjected antisense oligodeoxyribonucleotides (oligos), complementary to the specific domains of snRNAs. These oligos inhibit splicing throughout the different steps of the spliceosomal assembly Zillmann et al, 1988;Lamond et al, 1989). This was an alternative approach to generate the "frozen" prespliceosomal complexes on endogenous pre-mRNAs and follow the changes of the speckles.…”

A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

“…However, essentially no catalysis of the first step of splicing, 5' cleavage and lariat formation, occurred in these NS1 55S complexes. Consequently, although the binding of these five snRNPs to a splicing precursor is apparently required for splicing (2,8,11,14,15,33), our results indicate that this binding is not sufficient and that some additional step(s) needs to occur subsequent to this binding. One possibility suggested by recent data is that catalysis actually occurs concomitantly with the dissociation of the snRNPcontaining complexes.…”

“…The same snRNPs have been identified in the 50S to 60S spliceosomes by other methods (8,14,15,33). The association of the Ul snRNPs with the 50S to 60S spliceosomes appears to be more labile than that of the other snRNPs (2,11,33 interact with the 5' splice site and intron branch point, respectively. The 50S to 60S spliceosomes contain the products of the first step in splicing, 5' exon and lariat-3' exon, indicating that these spliceosomes are functional structures (1,4,7,10,24).…”

“…Analysis of the snRNP composition of 50S to 60S spliceosomes by affinity selection of biotinylated pre-mRNA on streptavidin-agarose beads indicated that the Ul, U2, U4, U5, and U6 snRNPs are present in these spliceosomes (2,11). The same snRNPs have been identified in the 50S to 60S spliceosomes by other methods (8,14,15,33). The association of the Ul snRNPs with the 50S to 60S spliceosomes appears to be more labile than that of the other snRNPs (2,11,33).…”

“…The same snRNPs have been identified in the 50S to 60S spliceosomes by other methods (8,14,15,33). The association of the Ul snRNPs with the 50S to 60S spliceosomes appears to be more labile than that of the other snRNPs (2,11,33). Of the five snRNPs found in spliceosomes, at least four, the Ul, U2, U4, and U6 snRNPs, have been shown to be required for splicing ( interact with the 5' splice site and intron branch point, respectively.…”