Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Widespread alternative and aberrant splicing revealed by lariat sequencing.

Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures.

Antibodies to repeat-containing antigens in Plasmodium falciparum are exposure-dependent and short-lived in children in natural malaria infections.

Protection against Plasmodium falciparum, which is primarily antibody-mediated, requires recurrent exposure to develop. The study of both naturally acquired limited immunity and vaccine induced protection against malaria remains critical for ongoing eradication efforts. Towards this goal, we deployed a customized P. falciparum PhIP-seq T7 phage display library containing 238,068 tiled 62-amino acid peptides, covering all known coding regions, including antigenic variants, to systematically profile antibody targets in 198 Ugandan children and adults from high and moderate transmission settings. Repeat elements - short amino acid sequences repeated within a protein - were significantly enriched in antibody targets. While breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, no such differences were observed for peptides without repeats, suggesting that antibody responses to repeat-containing regions may be more exposure dependent and/or less durable in children than responses to regions without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, potentially representing cross-reactive epitopes. PfEMP1 shared motifs with the greatest number of other antigens, partly driven by the diversity of PfEMP1 sequences. These data suggest that the large number of repeat elements and potential cross-reactive epitopes found within antigenic regions of P. falciparum could contribute to the inefficient nature of malaria immunity.