RNA splicing: unexpected spliceosome diversity.

A novel form of spliceosome, containing the minor snRNPs U11 and U12, splices a class of pre-mRNA introns with non-consensus splice sites. This unexpected spliceosome diversity has interesting implications for the evolution and expression of eukaryotic genes.

Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli.

Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.

Alternative 3' splice-site selection using HeLa cell nuclear extracts prepared with high-ionic buffers.

To investigate the role of cellular trans-acting factors in alternative 3' splice-site selection, a series of HeLa cell nuclear extracts were generated with salt washes ranging from 0.4 to 0.8 M salt. These extracts were tested with human beta-globin pre-mRNAs containing tandem 5' or 3' splice-site duplications as the substrates. High-salt (0.6 M and higher)-based buffers generated nuclear extracts that differentially processed pre-mRNAs containing competing 3' splice sites. High-salt extracts increased the usage of the distal 3' splice site, whereas no shift in 5' splice-site usage could be detected. Western analysis suggested that this shift in alternative 3' splice-site selection was not due to changes in the U2 snRNP auxiliary factor or polypyrimidine tract-binding protein levels.

Control of adenovirus alternative RNA splicing: effect of viral DNA replication on RNA splice site choice.

The primary transcripts of most adenovirus transcription units are processed into multiple, alternatively spliced mRNAs. The relative concentrations of such differentially processed mRNAs changes during the infectious cycle. The factors that control this temporal shift in mRNA abundance have not yet been characterized. In the experiments presented here we have examined mRNA synthesis from three viral transcription units: two early regions E1a and E1b, and late region L1. We show that viral DNA replication plays a key role in the control of cytoplasmic mRNA expression from these regions. In the absence of efficient late protein synthesis, viral DNA replication was sufficient to induce a substantial fraction of the E1a, E1b and L1 transcripts to shift from the early to the late pattern of mRNA structure. The shift was not complete under the conditions used, suggesting that viral proteins, although not essential for the process, play an important regulatory role. The requirement for late viral protein synthesis differed between the three transcription units examined. This dependence was most pronounced for correct L1 mRNA production. Viral DNA replication was sufficient to trigger a significant shift in L1 alternative 3' splice site selection. However, in the absence of late translation the L1 pre-mRNA was aberrantly spliced.

Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity.

We have purified a form of protein phosphatase 1 (PP1) from HeLa cell nuclei, in which the phosphatase is complexed to a regulatory subunit termed p99. We report here the cloning and characterisation of the p99 component. p99 mRNA is widely expressed in human tissues and immunofluorescence analysis with anti-p99 antibodies showed a punctate nucleoplasmic staining with additional accumulations within the nucleolus. The C-terminus of p99 contains seven RGG RNA-binding motifs, followed by eleven decapeptide repeats containing six or more of the following conserved residues (GHRPHEGPGG), and finally a putative zinc finger domain. Recombinant p99 suppresses the phosphorylase phosphatase activity of PP1 by > 90% and the canonical PP1-binding motif on p99 (residues 396-401) is unusual in that the phenylalanine residue is replaced by tryptophan.

Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation.

The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.

Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.

Evidence for a HeLa cell splicing activity that is necessary for activation of a regulated adenovirus 3' splice site.

Adenovirus late region 1 pre-mRNA splicing is temporally regulated during a lytic infection at the level of alternative 3' splice site usage to produce two mRNAs; the 52,55K and IIIa mRNAs which utilize the proximal and distal 3' splice sites, respectively. In vivo, the 52,55K mRNA is produced both early and late after infection, while IIIa is produced exclusively late in infection. Uninfected HeLa cell nuclear extracts, prepared with a low salt (0.4-0.5 M) or high salt (0.6 M and higher) wash, differed in their ability to splice 52,55K, IIIa and beta-globin transcripts. 52,55K and beta-globin precursors were spliced with similar efficiency in the low and high salt extract, while the IIIa mRNA was generated only in the high salt extract. Using the beta-globin pre-mRNA, no kinetic differences between the two types of extracts were observed. Nor were there any significant differences in the snRNA composition. The IIIa splicing activity did not appear to correlate with U2AF and pPTB levels. Our results suggest that a cellular trans-acting factor(s), which is required for adenovirus IIIa 3' splice site activation, is solubilized only at high salt concentrations.

Splicing of two weak 3' splice sites from the adenovirus major late region is enhanced in nuclear extracts from adenovirus infected cells.

The adenovirus major late transcription unit (MLTU) is an example of a complex alternatively spliced gene, in which more than 15 different 3' splice sites can be joined to a common 5' splice site. Maturation of the full repertoire of possible mRNAs requires late viral protein synthesis and occurs only at late stages of the infectious cycle (16-24 hpi). We are trying to decipher the mechanisms regulating alternative 3' splice site choice during the infectious cycle. Therefore, we examined the splicing activity of several 3' splice sites from the MLTU in vitro in nuclear extracts prepared from adenovirus infected cells (Ad NE) and from uninfected cells. The results suggest that pre-mRNAs with "weak" 3' splice sites (short, atypical polypyrimidine tracts) are activated and pre-mRNAs with long, prototypical polypyrimidine tracts are repressed in Ad NE. In fact, our data show a reciprocal correlation between the strength of a polypyrimidine tract, defined by its affinity for U2AF65K in vitro, and the efficiency of splicing in Ad NE. We are currently investigating the possible mechanisms responsible for this observed shift in 3' splice site choice during an adenovirus infection.

Role of the branch site/3'-splice site region in adenovirus-2 E1A pre-mRNA alternative splicing: evidence for 5'- and 3'-splice site co-operation.

The adenovirus E1A gene encodes five overlapping mRNAs which are processed by alternative RNA splicing from a common pre-mRNA. To characterize cis-acting sequence elements which are of importance for the alternative 5'-splice site selection deletion and substitution mutants within the intron that is common to all E1A mRNAs were constructed. Deletion of the wild-type E1A branch site/polypyrimidine tract resulted in activation of a functionally redundant sequence located within an A/T rich sequence just upstream of the normal E1A lariat branch site. Removal of both regulatory sequences abolished in vivo splicing completely and did not lead to activation of cryptic 3'-splice sites at other locations in the E1A pre-mRNA. Furthermore we show that the sequence around the E1A branch site/3'-splice site region may have a more direct effect on the efficiency by which the alternative E1A 5'-splice sites are selected. Replacing the E1A branch site/3'-splice site region with the corresponding sequence from the second intron of the rabbit beta-globin gene or the first intron of the major late transcription unit resulted in drastic changes in E1A 5'-splice site selection. For example, with the E1A/beta-globin hybrid gene the 9S mRNA became the most abundant E1A mRNA to accumulate. This contrasts with the wild-type E1A gene in which almost undetectable levels of 9S mRNA were produced in transient expression assays. Our results strongly suggest that a cooperative interaction between 5'- and 3'-splice sites on a pre-mRNA determines the outcome of alternative splicing.

Enhanced splicing of nonconsensus 3' splice sites late during adenovirus infection.

The adenovirus major late transcription unit is an example of an alternatively spliced gene, in which a common 5' splice site can be spliced to more than 15 different 3' splice sites. Here we show that the specificity in 3' splice site recognition changes during virus infection, such that 3' splice sites with long consensus-type polypyrimidine tracts are repressed while 3' splice sites with short atypical polypyrimidine tracts, which bind U2AF65K inefficiently, are enhanced in splicing in late virus-infected nuclear extracts. On the basis of these experiments, we discuss a mechanism that helps to explain how the complex pattern of major late mRNAs is produced late during virus infection.

Dynamic interactions between splicing snRNPs, coiled bodies and nucleoli revealed using snRNP protein fusions to the green fluorescent protein.

The U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) are subunits of splicing complexes that remove introns from mRNA precursors. snRNPs show a complex, transcription-dependent localization pattern in the nucleoplasm of mammalian cells that results from their association with several distinct subnuclear structures, including interchromatin granule clusters, perichromatin fibrils, and coiled bodies. Here we report the analysis of snRNP localization and interaction with the coiled body in live human cells using fusions of snRNP proteins and p80 coilin to the Green Fluorescent Protein (GFP). Despite the large size of the GFP tag, GFP fusions to both the core snRNP SmE and U1 specific U1A proteins assemble into snRNP particles and give an identical nuclear localization pattern to their endogenous counterparts. GFP-coilin localizes specifically to coiled bodies in a transcription-dependent fashion and provides an accurate marker for coiled bodies in a variety of human cell lines. Treatment of cells with the selective ser/thr-protein phosphatase inhibitor, okadaic acid, causes both GFP-snRNP and GFP-coilin proteins to accumulate within nucleoli, but does not result in nucleolar accumulation of the GFP-fused non-snRNP protein splicing factor ASF/SF2. In all four human cell lines tested, expression of a GFP-fused p80 coilin mutant with a single serine to aspartate substitution also caused nucleolar accumulation of splicing snRNPs and coilin, but not ASF/SF2, in structures resembling coiled bodies when viewed by electron microscopy. This work establishes an experimental system for analyzing snRNP trafficking in living cells and provides evidence that a reversible protein phosphorylation mechanism is involved in regulating interaction of snRNPs and coiled bodies with the nucleolus.

A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.

Two novel adenovirus-2 early region 1A mRNAs, designated 10S and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non-essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the E1A cDNA mutants it was possible to localize two functional domains in the E1A proteins; one region required for transcriptional activation (amino acids 140-185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27-98).