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Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5' side of a nucleotide of interest. Next, 32P is substituted for the phosphate at the 5' end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is approximately 90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.

Small nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus.

Mouse monoclonal antibodies (MAbs) were generated against Escherichia coli-produced U1snRNP-A (U1A) protein. U1A-specific MAbs as well as MAbs that reacted with both U1A and U2snRNP-B" (U2B") were isolated. MAb 12E12 was unique among the characterized MAbs because it failed to immunoprecipitate U1A protein produced by in vitro transcription and translation using rabbit reticulocyte lysates. However, when U1A protein was made using a wheat germ extract, MAb 12E12 could immunoprecipitate U1A quite readily, as did the other MAbs. These data suggest that the MAb 12E12 epitope is masked when U1A is prepared in reticulocyte lysate. Further studies showed that MAb 12E12 recognizes an epitope that is masked when U1A protein is bound to U1 RNA. The unique nature of MAb 12E12 was used to demonstrate that U1A could be immunoprecipitated from whole-cell extracts in a form that was free of U1 RNA and other snRNP components. However, this snRNP-free U1A (SF-A) was found to co-immunoprecipitate with a unique set of non-snRNP proteins. In order to confirm that U1A exists in at least two distinct complexes (snRNP bound and snRNP free), [35S]-labeled nucleoplasmic extracts were analyzed by sucrose density gradient fractionation and immunoprecipitation. MAb 12E12 specifically immunoprecipitated SF-A, which migrated in a novel non-snRNP complex. Specifically, proteins of approximately 58, 59, 63, 65, and 105 kDa co-sedimented and co-immunoprecipitated with SF-A. Our data show that a significant portion of the cellular U1A (at least 3% or approximately 30,000 molecules) exists in the nucleoplasm in one or more novel complexes. Our previous studies have demonstrated an effect of purified U1A on polyadenylation of pre-mRNAs and, consistent with this finding, purified antibodies to SF-A significantly diminish polyadenylation in vitro.

A minor class of introns with noncanonical splice sites has been identified in both vertebrate and invertebrate genomes. The divergent consensus sequences within these introns suggest that splicing might be via a mechanism distinct from that used by the major class of introns. The low abundance U12 snRNA has been proposed to base pair with the predicted branch site sequence of these minor class introns, probably bulging out an adenosine to act as the nucleophile in the first step of splicing. We have identified homologues of the previously characterized human U12 snRNA in both mouse and chicken, where the minor class of introns has also been found. The U12 sequences that potentially base pair with the putative branch site are invariant. Additional conserved sequences at the 5' end of U12 snRNA could dynamically base pair with U6 snRNA sequences flanking the hexanucleotide ACAGAG to form structures analogous to those of three U2-U6 interactions genetically defined as important in the major class of spliceosome. We have also isolated two human U12 snRNA genes. One gene is functional for transcription of U12 snRNA, whereas the other appears to be a pseudogene. Sequences of the 3' box in both U12 snRNA genes are strikingly similar and bear high resemblance to those of U1 and U2 genes. Upstream elements, including the PSE and the DSE, have been identified and characterized in the functional gene. These features indicate that transcription of U12 snRNA is driven by RNA polymerase II.

Psoralen cross-linking experiments in HeLa cell nuclear extracts have revealed the binding of U1 snRNA to substrates containing the SV40 late and adenovirus L3 polyadenylation signals. The sites of U1 cross-linking to the substrates map different distances upstream of the AAUAAA sequence to regions with limited complementarity to the 5' end of U1 snRNA. U1 cross-linking to the same site in the SV40 late pre-mRNA is enhanced by the addition of an upstream 3' splice site, which also enhances polyadenylation. Examination of different nuclear extracts reveals a correlation between U1 cross-linking and the coupling of splicing and polyadenylation, suggesting that the U1 snRNP participates in the coordination of these two RNA-processing events. Mutational analyses demonstrate that U1/substrate association cannot be too strong for coupling to occur and suggest that the U1 snRNP plays a similar role in recognition of internal and 3' terminal exons. Possible mechanisms for communication between the splicing and polyadenylation machineries are discussed, as well as how interaction of the U1 snRNP with 3' terminal exons might contribute to mRNA export.

To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box. These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract. The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events. Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al. 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect. The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs. The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.