Molecular principles underlying dual RNA specificity in the Drosophila SNF protein

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A′ protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A′ or U2 stem-loop IV and U2A′, SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A′ immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A′ can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts

evolution, structure and function of metazoan splicing factor PRPF39

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer

Induction of defences and within-plant variation on palatability in two brown algae from the northern-central coast of Chile: effects of mesograzers and UV radiation

Macroalgae possess different defense mechanisms in response to herbivory. Some species produce anti-herbivore secondary metabolites, but production of these substances can be costly. Therefore, algae may produce defensive metabolites only in response to herbivory (inducible defense) or defend particular parts of the alga differentially (within-alga variation). In the present study, we examined whether two species of brown algae from the SE-Pacific show evidence of inducible chemical defense (non-polar compounds) or within-alga variation of defense, which we estimated in form of palatability of differently treated algae to amphipod grazers (with live algae and agar-based food containing non-polar algal extracts). In Glossophora kunthii (C. Agardh) J. Agardh, we observed an increase in palatability after algae were acclimated for 12 days without grazers. Subsequent addition of grazers for 12 days then resulted in a reduction of palatability indicating the existence of inducible defense. After removal of grazers for 12 days, these induced effects again disappeared. The reaction of G. kunthii was triggered even by the mere presence of grazers, which suggests that this alga can respond to waterborne cues by reducing palatability. Effects were only found for agar-based food containing non-polar extracts, but not for live algae, suggesting that some parts of the algae are undefended. Our second experiment on within-alga variation confirmed that only apical (growth region) and basal parts (near the holdfast region) of G. kunthii are defended against herbivores. For the second species, Macrocystis integrifolia Bory, the first experiment revealed no induction of defense, while the second experiment on within-alga variation showed that amphipods avoided basal parts and in particular stipes of M. integrifolia but only in live algae. Although both studied algal species differed substantially in their defensive strategies, their reaction was independent of the presence or absence of UV radiation. Thus, it appears that UV effects play only a minor role in anti-herbivore defense, which is in accordance with most previous studies

Conformation-dependent ligand hot spots in the spliceosomal RNA helicase BRR2

The conversion of hits to leads in drug discovery involves the elaboration of chemical core structures to increase their potency. In fragment-based drug discovery, low-molecular-weight compounds are tested for protein binding and are subsequently modified, with the tacit assumption that the binding mode of the original hit will be conserved among the derivatives. However, deviations from binding mode conservation are rather frequently observed, but potential causes of these alterations remain incompletely understood. Here, two crystal forms of the spliceosomal RNA helicase BRR2 were employed as a test case to explore the consequences of conformational changes in the target protein on the binding behaviour of fragment derivatives. The initial fragment, sulfaguanidine, bound at the interface between the two helicase cassettes of BRR2 in one crystal form. Second-generation compounds devised by structure-guided docking were probed for their binding to BRR2 in a second crystal form, in which the original fragment-binding site was altered due to a conformational change. While some of the second-generation compounds retained binding to parts of the original site, others changed to different binding pockets of the protein. A structural bioinformatics analysis revealed that the fragment-binding sites correspond to predicted binding hot spots, which strongly depend on the protein conformation. This case study offers an example of extensive binding-mode changes during hit derivatization, which are likely to occur as a consequence of multiple binding hot spots, some of which are sensitive to the flexibility of the protein

Recruitment of a splicing factor to the nuclear lamina for its inactivation

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2–PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast

Effect of mesograzer and nutrient levels on the induction of defenses in several Brazilian acroalgae

Herbivory can greatly modify benthic community structure by affecting the distribution of algal species. To deter herbivores, algae have developed several mechanisms, including the induction of chemical and morphological defenses, which may be influenced by nutrient availability. We tested 4 red (Chondrophycus flagellifera, Cryptonemia seminervis, Osmundaria obtusiloba, Pterocladiella capillacea), 4 brown (Dictyota menstrualis, Lobophora variegata, Sargassum vulgare, Stypopodium zonale), and 1 green (Codium decorticatum) algae for inducible defenses following exposure to direct consumption by an amphipod community dominated by Elasmopus brasiliensis. In addition, the effects of water-borne cues from nearby grazed conspecifics and non-grazing consumers on the induction of defenses were examined in C. decorticatum under natural and enhanced (200% natural) nutrient levels. Induction of defense was assessed in choice-feeding assays, using live algae or artificial food containing non-polar extracts of amphipod-exposed (treated) and non-exposed (control) algae. Palatability levels, estimated as the relative difference in wet mass due to consumption in feeding assays between grazer-exposed and control plants, declined significantly in 3 species after the acclimatization period. Tissue from the directly consumed red alga P. capillacea (live alga) was significantly less palatable than tissue from the control plants. Likewise, a significant effect was observed in the brown alga L. variegata. Similar, although not statistically significant, trends were observed in 6 other species. For the green alga C. decorticatum, nutrient enrichment did not affect induction of defenses by herbivores, yet unfertilized plants were more palatable than fertilized conspecifics

The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4’s intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling

A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing