The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.

An in vitro reconstitution/splicing complementation system has been developed which has allowed the investigation of the role of mammalian U2 and U5 snRNP components in splicing. U2 or U5 snRNP cores are first reconstituted from purified native snRNP core proteins and snRNA in the absence of cellular extract and are subsequently added to splicing extracts depleted of either U2 or U5 snRNP. When snRNPs reconstituted with HeLa U2 or U5 snRNA were added to U2‐ or U5‐depleted nuclear extract, splicing was complemented. Addition of naked snRNA, on the other hand, did not restore splicing, demonstrating that the core proteins are essential for both U2 and U5 snRNP functions in splicing. Hybrid U2 or U5 snRNPs, reconstituted with core proteins isolated from U1 or U2 snRNPs, were equally active in splicing complementation, indicating that the snRNP core proteins are functionally interchangeable. U5 snRNPs reconstituted from in vitro transcribed U5 snRNA restored splicing to a level identical to that observed with particles reconstituted from authentic HeLa U5 snRNA. In contrast, splicing could not be restored to U2‐depleted extract by the addition of snRNPs reconstituted from synthetic U2 snRNA, suggesting that U2 snRNA base modifications are essential for U2 snRNP function.

The origin of the intervening sequences (introns), which are removed during RNA maturation, is currently unknown. They are found in most genes encoding messenger RNAs, but are lacking in almost all small nuclear (sn)RNAs. One exceptional snRNA (U6) is part of the spliceosomal machinery that is involved in messenger RNA maturation. It has been suggested that its intron arose as a result of incorrect splicing of a messenger RNA precursor. This study revealed the presence of an intron, with the characteristic features of nuclear introns from precursors to messenger RNA, in the two genes coding for Saccharomyces cerevisiae U3 snRNA. The branch point was GACTAAC instead of the TACTAAC sequence found in all yeast introns examined so far. As U3 is a nucleolar snRNA required for maturation of ribosomal RNA, its intron could not have been acquired from aberrant messenger RNA processing in a spliceosome.

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5 and 3 halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.Nuclear pre-mRNA splicing proceeds via a two-step mechanism. In the first step, the pre-mRNA is hydrolyzed at the 5Ј splice site and the 5Ј end of the intron interacts concomitantly with an adenosine at the so-called branch point. The splicing intermediates thus generated include exon 1 and a lariat structure comprised of the intron and exon 2. In the second step, hydrolysis at the 3Ј splice site and the concomitant ligation of exons 1 and 2 give rise to the mRNA and the excised intron in the form of a lariat. Both reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex formed by the ordered interaction of numerous splicing factors and the four small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, and U4/U6, with conserved regions of the pre-mRNA (reviewed in references 19, 27, and 34). Spliceosome assembly is initiated by the interaction of the U1 and U2 snRNPs with the 5Ј splice site and branch site, respectively, thereby generating the so-called prespliceosome, or complex A. Mature spliceosomes (i.e., complexes B and C) are ultimately formed by the subsequent interaction of the U4/U6 and U5 snRNPs, in the form of a preassembled U4/U6.U5 tri-snRNP complex (reviewed in references 19 and 34).The assembly of a catalytically active spliceosome requires the formation of a network of RNA-RNA interactions which favorably position the chemically reactive groups of the premRNA for catalysis (for reviews, see references 26 and 38). The U5 snRNP has been proposed to play a central role in recognizing and aligning the 5Ј and 3Ј splice sites for catalysis, and its function appears to be mediated, at least in part, by base pairing interactions between the U5 small nuclear RNA (snRNA) ...

The conformation of Saccharomyces cerevisiae U3 snRNA (snR17A RNA) in solution was studied using enzymatic and chemical probes. In vitro synthesized and authentic snR17A RNAs have a similar conformation in solution. The S. cerevisiae U3 snRNA is folded in two distinct domains. The 5'-domain has a low degree of compactness; it is constituted of two stem-loop structures separated by a single-stranded segment, which has recently been proposed to basepair with the 5'-ETS of pre-ribosomal RNA. We demonstrate that, as previously proposed, the 5'-terminal region of U3 snRNA has a different structure in higher and lower eukaryotes and that this may be related to pre-rRNA 5'-ETS evolution. The S. cerevisiae U3 snRNA 3'-domain has a cruciform secondary structure and a compact conformation resulting from an higher order structure involving the single-stranded segments at the center of the cross and the bottom parts of helices. Compared to tRNA, where long range interactions take place between terminal loops, this represents another kind of tertiary folding of RNA molecules that will deserve further investigation, especially since the implicated single-strands have highly evolutionarily conserved primary structures that are involved in snRNP protein binding.