Ursula Ryder

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy‐dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS‐binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino‐terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1‐like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino‐terminal domain inhibits import of NLS‐containing proteins when added to an in vitro nuclear transport assay. While full‐length hSRP alpha is able to leave the nucleus, the amino‐terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS‐containing substrates to the protein import machinery.

Identification of small molecule inhibitors of pre-mRNA splicing.

Background: There is a need for new small molecule pre-mRNA splicing inhibitors as biotools. Results: High throughput screening resulted in the identification of small molecule splicing inhibitors that are active in vitro and in cells. Conclusion: New small molecules for studying pre-mRNA splicing in vitro and in cells are identified. Significance: Small drug-like molecules are identified that modulate splicing in vitro and in cells. Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed “madrasin,” i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.

Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA.

Biotinylated 2′‐OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP‐‐pre‐mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre‐mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre‐mRNA is blocked by the pre‐binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5′ terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2′‐OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre‐mRNA are precursors to functional spliceosomes.

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection, in pre-mRNA splicing

Human factor C1 (HCF‐1) is needed for the expression of herpes simplex virus 1 (HSV‐1) immediate‐early genes in infected mammalian cells. Here, we provide evidence that HCF‐1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF‐1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF‐1 in the BHK21 hamster cell line tsBN67, at the non‐permissive temperature, inhibits the proteins interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild‐type HCF‐1 in tsBN67 cells restores splicing at the non‐permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature‐sensitive cell cycle arrest phenotype, suggesting that HCF‐1‐dependent splicing events may be required for cell cycle progression.

Exo70, a subunit of the exocyst complex, interacts with SNEV hPrp19/hPso4 and is involved in pre-mRNA splicing

The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin-proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEV(hPrp19/hPso4) and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.

Novel solid-phase synthesis of branched oligoribonucleotides, including a substrate for the RNA debranching enzyme

An effective new route for synthesizing branched oligoribonucleotides in the solid phase in the 5′ to 3′ direction has been developed. This required the synthesis of reversed monomers, viz. protected nucleoside 5′-phosphoramidites bearing 2′-O-Fpmp and (3′-O-pixy) protecting groups as well as special branch-point monomers, viz. protected nucleoside 5′-phosphoramidites bearing either 2′, 3′-O-dipixyl protection in the case of adenosine, cytidine and uridine, or 2′,3′-O-dilaevulinyl protection in the case of guanosine. These monomers are assembled on commercial synthesizers into branched oligoribonucleotides in high yield, the crude products are readily purified by reversedphase HPLC whilst still partially protected, and the fully deprotected products are conveniently analysed by electrospray mass spectrometry. Moreover, the branched oligoribonucleotides can be recognised and cleaved by a specific 2′–5′ phosphodiesterase present in mammalian cell nuclei. We expect that this will prove valuable for future biochemical and biological studies on the properties of branched RNA molecules and the protein factors and enzymes that interact with branched RNA substrates.

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.