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A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r‐protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre‐mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre‐mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.

By cross-hybridization with a cDNA probe for the Xenopus laevis ribosomal protein L1 we have been able to isolate the homologous genes from a Saccharomyces cerevisiae genomic library. We have shown that these genes code for a ribosomal protein which was previously named L2. In yeast, like in X. laevis, these genes are present in two copies per haploid genome and, unlike the vertebrate counterpart, they do not contain introns. Amino acid comparison of the X. laevis L1 and S. cerevisiae L2 proteins has shown the presence of a highly conserved protein domain embedded in very divergent sequences. Although these sequences are very poorly homologous, they confer an overall secondary structure and folding highly conserved in the two species.

Cloned gene for Xenopus ribosomal protein L1 was injected into fertilized eggs, and its expression was analyzed during the period of embryo development when the mRNAs produced by the endogenous ribosomal protein genes are still silent due to a translational control. The injected genes replicated extensively, and a 10-fold excess of L1 mature transcript accumulated in the embryo. This was accompanied by a small amount of incompletely processed L1 RNA that still contained one out of nine introns, a molecule never observed in normal conditions. The excess mature L1 mRNA was distributed between polysomes and messenger ribonucleoproteins (mRNPs) in the same relative proportion observed in control embryos of the same stage. Therefore, more L1 mRNA was loaded onto polysomes and caused the appearance of L1 protein when this was not yet detectable in control embryos. The results suggest a relationship between the excess amount of L1 protein and the alteration in processing of its transcripts.

A specific control regulates, at the level of RNA splicing, the expression of the L1 ribosomal protein gene in Xenopus laevis. Under particular conditions, which can be summarized as an excess of free L1 protein, a precursor RNA which still contains two of the nine introns of the L1 gene accumulates. In addition to the splicing block the two intron regions undergo specific endonucleolytic cleavages which produce abortive truncated molecules. The accumulation of mature L1 RNA therefore results from the regulation of the nuclear stability of its precursor RNA. We propose that a block to splicing can permit the attack of specific intron regions by nucleases which destabilize the pre‐mRNA in the nucleus. Therefore the efficiency of splicing could indirectly control the stability of the pre‐mRNA.

Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5′ end of this gene has been mapped within a 20‐pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60‐nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post‐transcriptional level and it involves the block of the normal splicing of some intron sequences.

The ribosomal protein L2 (rpL2) of Saccharomyces cerevisiae regulates the accumulation of its own mRNA by a feedback mechanism. An RNA sequence is responsible for this control, initially characterized as a 360 nucleotide‐long region, localized at the 5′ end of the transcript. This region, fused to an unrelated coding sequence, is able to down‐regulate the accumulation of the chimeric transcript when increased levels of rpL2 are induced in the cell. The target regulatory region also responds to regulation when inserted inside an intron, demonstrating that the control process can take place inside the nucleus. Deletion analysis from the 5′ and 3′ borders have restricted the responsive region to approximately 200 nt. The insertion of a poly‐G cassette downstream of the regulatory region allowed the identification of truncated 3′ cut‐off poly(A)+ RNA molecules. The parallel identification of cut‐off molecules containing the 5′ portion of the transcript allowed us to deduce that the truncated products originate by endonucleolytic cleavage. Altogether, these results are consistent with a mechanism by which the presence of excess amounts of rpL2 in the cell triggers its own mRNA to a degradative pathway; this involves an initial endonucleolytic cleavage that is followed by exonucleolytic trimming. Such a regulatory mechanism shows interesting analogies with the translational regulation of r‐proteins in Escherichia coli.

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14.

Splicing of the regulated third intron of the L1 ribosomal protein gene of Xenopus laevis has been studied in vivo by oocyte microinjection of wild-type and mutant SP6 precursor RNAs and in vitro in the heterologous HeLa nuclear extract. We show that two different phenomena combine to produce the peculiar splicing phenotype of this intron. One, which can be defined constitutive, shows the same features in the two systems and leads to the accumulation of spliced mRNA, but in very small amounts. The low efficiency of splicing is due to the presence of a noncanonical 5' splice site which acts in conjunction with sequences present in the 3' portion of the intron. The second leads to the massive conversion of the pre-mRNA into site specific truncated molecules. This has the effect of decreasing the concentration of the pre-mRNA available for splicing. We show that this aberrant cleavage activity occurs only in the in vivo oocyte system and depends on the presence of an intact U1 RNA.

The gene encoding ribosomal protein L1 in Xenopus laevis is known to be posttranscriptionally regulated; the third intron can be processed from the pre-mRNA in two alternative ways, resulting either in the production of L1 mRNA or in the release of a small nucleolar RNA (U16). The formation of splicing complexes was studied in vivo by oocyte microinjection. We show that spliceosome assembly is impaired on the L1 third intron and that the low efficiency of the process is due to the presence of suboptimal consensus sequences. An analysis of heterogeneous nuclear ribonucleoprotein (hnRNP) distribution was also performed, revealing a distinct site for hnRNP C binding proximal to the 5' end of the L1 third intron. Cleavage, leading to the production of the small nucleolar RNA U16, occurs in the same position, and we show that conditions under which hnRNP C binding is reduced result in an increase of the processing activity of the intron.

The expression of the yeast L2 r‐protein gene is controlled at the level of mRNA accumulation. The product of the gene appears to participate in this regulation by an autogenous feedback mechanism. This control does not operate at the level of transcription but instead affects L2 mRNA accumulation. This autogenous regulation of mRNA accumulation provides an interesting analogy to the autogenous translational regulation of r‐proteins in Escherichia coli.

We report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre‐mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre‐mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components.

It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

We have shown that the accumulation of mature L1 mRNA is regulated, by reducing the efficiency of the splicing reaction, at the level of nuclear RNA stability. The present analysis is directed to the identification of the factors that are responsible for the block of splicing and for the specific cleavage of the precursor RNA. The results obtained so far demonstrated that a protein of 40 K interacts specifically with introns 2 and 3.