Biomedical subjects

Ruth Sperling

Publications and source records attributed to Ruth Sperling.

6 recordsLinked to original sources

PubMed2004

Regulation of splicing: the importance of being translatable.

RNA sequences that conform to the consensus sequence of 5' splice sites but are not used for splicing occur frequently in protein coding genes. Mutational analyses have shown that suppression of splicing at such latent sites may be dictated by the necessity to maintain an open reading frame in the mRNA. Here we show that stop codon frequency in introns having latent 5' splice sites is significantly greater than that of introns lacking such sites and significantly greater than the expected occurrence by chance alone. Both observations suggest the occurrence of a general mechanism that recognizes the mRNA reading frame in the context of pre-mRNA.

Humans↗

PubMed2003

Conserved sequence elements associated with exon skipping.

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5' and 3' splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.

Alternative Splicing↗

PubMed2002

Stop codons affect 5' splice site selection by surveillance of splicing.

Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5' SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5' SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs.

Alternative Splicing↗

PubMedDate not supplied

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine.

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.

Cell Nucleus Structures↗

PubMedDate not supplied

Conservation of an open-reading frame as an element affecting 5' splice site selection.

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.

Algorithms↗

PubMedDate not supplied

Large nuclear RNP particles--the nuclear pre-mRNA processing machine.

Processing of nuclear pre-mRNA is an important step in the regulation of gene expression and involves 5(')- and 3(')-end processing, splicing, and editing. Mammalian nuclear pre-mRNAs are assembled in large ribonucleoprotein (lnRNP) complexes, in which the entire population of nuclear pre-mRNA is individually packaged until it is exported to the cytoplasm. The lnRNP particles are supraspliceosomal complexes. They are composed of four spliceosomal substructures and an additional one, which are interconnected by the pre-mRNA, and have an overall mass of 21MDa. The additional substructure was proposed to harbor additional processing activities, such as editing components that were shown to be associated with the lnRNP particles. Here we show that the cap-binding proteins (CBPs), CBP20 and CBP80, are associated with the lnRNP particles, as well as components of the 3(')-end-processing activity. These results, together with our previous demonstration of the association of splicing factors and A-to-I editing enzymes with lnRNP particles, support the view that the lnRNP particles are the nuclear pre-mRNA processing machine. Such a machine is required to execute the nuclear processing steps of the pre-mRNA in an accurate and regulated manner. The supraspliceosomal pre-mRNA processing machine, in which each substructure represents a functional spliceosome, provides a frame onto which the pre-mRNA is folded. It allows juxtaposition of exons about to be spliced, while introns are looped out of each of the respective spliceosomes. This model can account for regulated alternative splicing, which is a major source of protein versatility in mammals.

Blotting, Western↗