Modulation of alternative splicing by expression of small nuclear ribonucleoprotein polypeptide N.

Alternative splicing of pre-mRNA, catalyzed by small nuclear ribonucleoproteins (snRNPs), plays an important role in proteome complexity and the modulation of cellular functions. snRNP polypeptide N (SmN), is tissue-specifically expressed, where it replaces snRNP polypeptide B (SmB)/B' in the Sm core assembly of snRNPs. Recent studies have demonstrated that perturbation of snRNPs leads to alternative splicing, but whether SmN modulates functions of the splicing machinery remains unclear. In this study, we found that ectopic expression of SmN increased utilization of the proximal 5' splice site on an adenovirus early gene 1A reporter. To evaluate the molecular mechanisms underlying SmN-dependent alternative splicing, we generated a HeLa cell line with an inducible expression system for SmN. Upon SmN induction, SmB/B' expression decreased dramatically, despite only small changes in the level and splicing pattern of SNRPB mRNA. In addition, SmN was incorporated into the U2 snRNP but not into the U1 snRNP after induction. Sedimentation analysis revealed a decrease in the level of mature U2 snRNP. This result suggests that SmN incorporation into the Sm core may impede processing, decreasing the level of functional U2 snRNP. We also found that the inclusion frequencies of alternatively spliced exons in the bridging integrator 1 and exocyst complex component 7 (EXOC7) genes were modulated by SmN expression. An enhanced GFP-EXOC7 reporter was used to confirm that SmN increases the inclusion frequency of EXOC7 exon 7. Taken together, our findings indicate that SmN expression reduces the level of mature U2 snRNP, leading to alternative splicing.

Exon, intron and splice site locations in the spliceosomal B complex.

In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5' and 3' exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The gold-labelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process.

Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells.

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B", and U2A ', nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.

Generation of a monoclonal antibody against the mouse Sf3b1 (SAP155) gene product for U2 snRNP component of spliceosome.

SAP155 is a subunit of the U2 snRNP, and plays an important role for prespliceosome assembly and splicing catalysis of the major spliceosome. Recently, it was reported that SAP155 was also a subunit of the minor spliceosome. These suggest that SAP155 is essential for the removal of any type of intron. More recently, a homolog of SAP155 cDNA, designated Sf3b1, was isolated from mouse. In this study, we report the generation of a monoclonal antibody (MAb) against murine Sf3b1 protein. This MAb recognizes endogenous Sf3b1 gene product by Western blotting, but less efficiently by immunoprecipitation.

Identification of both shared and distinct proteins in the major and minor spliceosomes.

In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the minor spliceosome and shed light on its evolutionary relationship to the major spliceosome.

Coiled body numbers in the Arabidopsis root epidermis are regulated by cell type, developmental stage and cell cycle parameters.

We have used whole mount immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B", and whole mount in situ hybridization with an anti-sense probe to a conserved region of U2 snRNA, in combination with confocal microscopy, to examine the organization of spliceosomal components throughout the development of the Arabidopsis thaliana root epidermis. We show that the number of coiled bodies, nuclear organelles in which splicing snRNPs and snRNAs concentrate, is developmentally regulated in the Arabidopsis root epidermis. Firstly, there is a progression from a small number of coiled bodies in the quiescent centre and initial cells, to a larger number in the cell division zone, returning to a lower number in the cell elongation and differentiation zone. Secondly, trichoblasts (root-hair forming epidermal cells) have on average 1.5 times more and often smaller coiled bodies than atrichoblasts (hairless epidermal cells). Moreover, we have shown that these differences in coiled body numbers are related to differences in cell cycle stage, cell type and developmental stage, but are not due to differences in nucleolar or general metabolic activity per se. We discuss possible explanations, including a model in which coiled bodies coalesce during interphase, for the developmental dynamics of coiled bodies.

snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes.

Splice site recognition and catalysis of the transesterification reactions in the spliceosome are accompanied by a dynamic series of interactions involving conserved or invariant sequences in the spliceosomal snRNAs. We have used site-specific photoactivated crosslinking in yeast spliceosomes to monitor interactions between snRNAs and exon sequences near the 5' and 3' splice sites. The last nucleotide of the 5' exon can be crosslinked to an invariant loop sequence in U5 SnRNA before and after 5' splice site cleavage. The first nucleotide of the 3' exon can also be crosslinked to the same U5 loop sequence, but this contact is only detectable after the first transesterification. These results are in close agreement with earlier data from mammalian splicing extracts, and they are consistent with a model in which U5 snRNA aligns the 5' and 3' exons for the second transesterification. After the first catalytic step of splicing, the first nucleotide of the 3' exon can also crosslink to nt U23 in U2 snRNA. This is one of a cluster of residues in U2-U6 helix I implicated by mutational analysis in the second catalytic step of splicing. The crosslinking data suggest that these residues in U2-U6 helix I are in close proximity to the scissile phosphodiester bond at the 3' splice site prior to the second transesterification. These results constitute the first biochemical evidence for a direct interaction between the 3' splice site and U2 snRNA.

Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes.

Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form. By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.