FUBP1 is a general splicing factor facilitating 3' splice site recognition and splicing of long introns.

Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.

Malaria Molecular Surveillance in the Peruvian Amazon with a Novel Highly Multiplexed Plasmodium falciparum AmpliSeq Assay.

Molecular surveillance for malaria has great potential to support national malaria control programs (NMCPs). To bridge the gap between research and implementation, several applications (use cases) have been identified to align research, technology development, and public health efforts. For implementation at NMCPs, there is an urgent need for feasible and cost-effective tools. We designed a new highly multiplexed deep sequencing assay (Pf AmpliSeq), which is compatible with benchtop sequencers, that allows high-accuracy sequencing with higher coverage and lower cost than whole-genome sequencing (WGS), targeting genomic regions of interest. The novelty of the assay is its high number of targets multiplexed into one easy workflow, combining population genetic markers with 13 nearly full-length resistance genes, which is applicable for many different use cases. We provide the first proof of principle for hrp2 and hrp3 deletion detection using amplicon sequencing. Initial sequence data processing can be performed automatically, and subsequent variant analysis requires minimal bioinformatic skills using any tabulated data analysis program. The assay was validated using a retrospective sample collection (n = 254) from the Peruvian Amazon between 2003 and 2018. By combining phenotypic markers and a within-country 28-single-nucleotide-polymorphism (SNP) barcode, we were able to distinguish different lineages with multiple resistance haplotypes (in dhfr, dhps, crt and mdr1) and hrp2 and hrp3 deletions, which have been increasing in recent years. We found no evidence to suggest the emergence of artemisinin (ART) resistance in Peru. These findings indicate a parasite population that is under drug pressure but is susceptible to current antimalarials and demonstrate the added value of a highly multiplexed molecular tool to inform malaria strategies and surveillance systems. IMPORTANCE While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance. In addition, there is a lack of standardized laboratory protocols and automated analysis pipelines to generate reproducible and timely results useful for relevant stakeholders. With our standardized laboratory and bioinformatic workflow, malaria genetic surveillance data can be readily generated by surveillance researchers and malaria control programs in countries of endemicity, increasing ownership and ensuring timely results for informed decision- and policy-making.

Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation.

Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1. This behavior resembled the 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.