Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair.

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.

The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity.

The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications. Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP. By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins. Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt. In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge. The peculiar KT-15 motif of P. abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p. To get more information on the P. abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution. In spite of their sequence divergence, the free P. abyssi and bound H. marismortui proteins were found to have highly similar structures. Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding. In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264. The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP.

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.

Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts.

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.

U3 snoRNA genes with and without intron in the Kluyveromyces genus: yeasts can accommodate great variations of the U3 snoRNA 3'-terminal domain.

The U3 snoRNA coding sequences from the genomic DNAs of Kluyveromyces delphensis and four variants of the Kluyveromyces marxianus species were cloned by PCR amplification. Nucleotide sequence analysis of the amplification products revealed a unique U3 snoRNA gene sequence in all the strains studied, except for K. marxianus var. fragilis. The K. marxianus U3 genes were intronless, whereas an intron similar to those of the Saccharomyces cerevisiae U3 genes was found in K. delphensis. Hence, U3 genes with and without intron are found in yeasts of the Saccharomycetoideae subfamily. The secondary structure of the K. delphensis pre-U3 snoRNA and of the K. marxianus mature snoRNAs were studied experimentally. They revealed a strong conservation in yeasts of (1) the architecture of U3 snoRNA introns, (2) the 5'-terminal domain of the mature snoRNA, and (3) the protein-anchoring regions of the U3 snoRNA 3' domain. In contrast, stem-loop structures 2, 3, and 4 of the 3' domain showed great variations in size, sequence, and structure. Using a genetic test, we show that, in spite of these variations, the Kluyveromyces U3 snoRNAs are functional in S. cerevisiae. We also show that S. cerevisiae U3A snoRNAs lacking the stem-loop structure 2 or 4 are functional. Hence, U3 snoRNA function can accommodate great variations of the RNA 3'-terminal domain.

Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency.

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor. This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome. The peculiar cruciform structure of the U3A snoRNA 3'-terminal region is formed in the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins. The intron forms a highly folded structure with a long central stem-loop structure that brings the 5' box and the branch site together. This is in agreement with the idea that secondary structure interactions are necessary for efficient splicing of long introns in yeast. The 3' splice site is in a bulged loop and the branch site sequence is single-stranded. Surprisingly, the 5' splice site is involved in a 6-base pair interaction. We used in vitro splicing experiments to show that, despite a noncanonical branch site sequence and a base paired 5' splice site, transcripts that mimic the authentic pre-U3A snoRNA are spliced very efficiently in vitro. Sequestering the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence. Analysis of spliceosomal complex formation revealed a cumulative negative effect of a base pair interaction at the 5' splice site and of a deviation to the consensus sequence at the branch site on the efficiency of spliceosome formation in vitro.

The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA.

The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA. Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required. We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3. By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H. wingei. As in the S. cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome. Consequently, this unusual property is not restricted to the yeast genus Saccharomyces. The introns of the H. wingei and S. cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region. The 3' domains of the H. wingei and S. cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures. The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved. The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H. wingei that restore 10 bp of perfect complementarity to the 5' ETS. This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction.

Secondary structure conservation of the U3 small nucleolar RNA introns in Saccharomyces.

Using a strategy based on PCR amplification of DNA and sequence analysis, we showed that the presence of introns with the characteristic features of introns spliced in a spliceosome, in the U3A and U3B snoRNA genes that code for the U3 small nucleolar RNA, is not a property restricted to Saccharomyces cerevisiae. It is probably an ancient property of yeasts from the genus Saccharomyces. We detected the U3A and U3B snoRNA genes in Saccharomyces bayanus and in a lager brewing yeast strain. The U3A and U3B intronic sequences are highly conserved. Two additional "U3B-like" snoRNA genes were detected in the lager brewing yeast. Their intronic sequences show several differences, when compared to the U3B intronic sequence. However, despite the numerous mutations, the intron secondary structure is conserved, especially, the central structure. This strongly suggests an important role of this central stem/loop structure for spliceosome assembly and efficient splicing.

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

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.

An experimental study of Saccharomyces cerevisiae U3 snRNA conformation in solution.

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.

An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae.

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.