The accumulation of mature RNA for the Xenopus laevis ribosomal protein L1 is controlled at the level of splicing and turnover of the precursor RNA (original) (raw)
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The EMBO Journal
A specific control regulates, at the level of RNA splicing, the expression of the Li ribosomal protein gene in Xenopus laevis. Under particular conditions, which can be summarized as an excess of free Li protein, a precursor RNA which still contains two of the nine introns of the Li 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 Li 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.
Splicing of the regulated third intron of the LI ribosomal protein gene ofXenopus 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 Ul RNA.
Molecular and cellular …, 1994
The gene encoding ribosomal protein LI 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 LI 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 Li 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 LI 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.
Nucleic Acids Research, 1992
Sequences corresponding to the third intron of the X.Iaevis LI ribosomal protein gene were isolated from the second copy of the X.Iaevis gene and from the single copy of X.tropicalis. Sequence comparison revealed that the three introns share an unusual sequence conservation which spans a region of 110 nucleotides. In addition, they have the same suboptimal 5' splice sites. The three introns show similar features upon oocyte microinjection: they have very low splicing efficiency and undergo the same site specific cleavages which lead to the accumulation of truncated molecules. Computer analysis and RNAse digestions have allowed to assigne to the conserved region a specific secondary structure. Mutational analysis has shown that this structure is important for conferring the cleavage phenotype to these three introns. Competition experiments show that the cleavage phenotype can be prevented by coinjection of excess amounts of homologous sequences.
In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis
Molecular and cellular biology, 1994
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 ...
Biochemical and Biophysical Research Communications, 1992
The splicing of the third intron of the LI r-protein gene of X./aevis was studied in the heterologous in vitro HeLa nuclear system. Despite the evolutionary distance, the cis-elements responsible for the default process play a similar role in the two organisms. Analysis of the splicing of various mutant substrates showed that the 5' splice site is primarily responsible for the low efficiency of splicing of the third intron. The suboptimal 5' splice site sequence leads to the utilization of an upstream alternative site which corresponds to the one utilized in vivo. The accumulation of splicing intermediates in the in vitro system allowed the identification of the branch site and of the branch consensus sequence. In contrast, the in vivo regulatory mechanism involving cleavage of the pre-mRNA is not mimicked in the HeLa extract.
The Journal of Cell Biology, 2000
Transcription and splicing of messenger RNAs are temporally and spatially coordinated through the recruitment by RNA polymerase II of processing factors. We questioned whether RNA polymerase I plays a role in the recruitment of the ribosomal RNA (rRNA) processing machinery. During Xenopus laevis embryogenesis, recruitment of the rRNA processing machinery to the nucleolar domain occurs in two steps: two types of precursor structures called prenucleolar bodies (PNBs) form independently throughout the nucleoplasm; and components of PNBs I (fibrillarin, nucleolin, and the U3 and U8 small nucleolar RNAs) fuse to the nucleolar domain before components of PNBs II (B23/NO38). This fusion process is independent of RNA polymerase I activity, as shown by actinomycin D treatment of embryos and by the lack of detectable RNA polymerase I at ribosomal gene loci during fusion. Instead, this process is concomitant with the targeting of maternally derived pre-rRNAs to the nucleolar domain. Absence of...
Journal of Molecular Biology, 1969
Polyacrylamide gel electrophoresis was used to analyse the rapidly labelled RNA in Xenopus Levis cultured kidney cells. The ribosomal precursor was identified by its base composition and found to have a molecular weight of 2.5 to 2.6 x 106. This is O-3 to 0.4 x lo6 greater than the sum of the weights of the ribosomal RNA. This excess of non-ribosomal RNA has a high content of G+C and is assumed to be lost during processing. The precursor in plant tissues was shown to be similar to that in Xenqnus. A relatively long-lived intermediate in the processing was found in Xenopus and plants, which had a molecular weight of 0.1 x lo6 greater than the heavy ribosomal RNA; it was assumed to be a precursor to the latter. These amounts of excess non-ribosomal RNA are much smaller than in the mammalian 45 and 32 s precursors. It is concluded that the very high molecular weight precursors which contain about 40% of excess RNA are peculiar to the mammals. The results are correlated with other work on the ribosomal DNA and on the structures of the nucleolar core.
Small nuclear RNA transcription and ribonucleoprotein assembly in early Xenopus development
The Journal of Cell Biology
The Xenopus egg and embryo, throughout the transcriptionally inactive early cleavage period, were found to contain a store of approximately 8 x 108 molecules of the small nuclear RNA (snRNA) U 1, sufficient for 4,000-8,000 nuclei. In addition, when transcription is activated at the twelfth cleavage (4,000 cell-stage), the snRNAs U1, U2, U4, U5, and U6 are major RNA polymerase II products. From the twelfth cleavage to gastrulation, U1 RNA increases sevenfold in 4 h, paralleling a similar increase in nuclear number. This level of snRNA transcription is much greater than that typical of somatic cells, implying a higher rate of U1 transcription or a greater number of U1 genes active in the embryo. The Xenopus egg also contains snRNP proteins, since it has the capacity to package exogenously added snRNA into immunoprecipitable snRNP particles, which resemble endogenous particles in both sedimentation coefficient and T1 RNase digestibility. SnRNP proteins may recognize conserved secondary structure of U1 snRNA since efficient packaging of both mouse and Drosophila U1 RNAs, differing 30% in sequence, occurs. The Xenopus egg and embryo can be used to pose a number of interesting questions about the transcription, assembly, and function of snRNA.