At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression.

Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5' and 3' ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5' and 3' ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.

Chromosomal instability by mutations in the novel minor spliceosome component CENATAC.

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.

Genetic diversity and population structure of Plasmodium falciparum in Kenyan-Ugandan border areas.

Kenya has, in the last decade, made tremendous progress in the fight against malaria. Nevertheless, continued surveillance of the genetic diversity and population structure of Plasmodium falciparum is required to refine malaria control and to adapt and improve elimination strategies. Twelve neutral microsatellite loci were genotyped in 201 P. falciparum isolates obtained from the Kenyan-Ugandan border (Busia) and from two inland malaria-endemic sites situated in western (Nyando) and coastal (Msambweni) Kenya. Analyses were done to assess the genetic diversity (allelic richness and expected heterozygosity, [He ]), multilocus linkage disequilibrium ( ISA ) and population structure. A similarly high degree of genetic diversity was observed among the three parasite populations surveyed (mean He  = 0.76; P > 0.05). Except in Msambweni, random association of microsatellite loci was observed, indicating high parasite out-breeding. Low to moderate genetic structure (FST  = 0.022-0.076; P < 0.0001) was observed with only 5% variance in allele frequencies observed among the populations. This study shows that the genetic diversity of P. falciparum populations at the Kenyan-Ugandan border is comparable to the parasite populations from inland Kenya. In addition, high genetic diversity, panmixia and weak population structure in this study highlight the fitness of Kenyan P. falciparum populations to successfully withstand malaria control interventions.

Le Kenya a réalisé d'énormes progrès au cours de la dernière décennie dans la lutte contre le paludisme. Néanmoins, une surveillance continue de la diversité génétique et de la structure de la population de P. falciparum est nécessaire pour affiner la lutte contre le paludisme et pour adapter et améliorer les stratégies d'élimination. Douze loci microsatellites neutres ont été génotypés chez 201 isolats de P. falciparum provenant de la frontière entre le Kenya et l'Ouganda (Busia) et de deux sites d'endémie palustre situés dans l'ouest (Nyando) et sur la côte (Msambweni), au Kenya. Des analyses ont été effectuées pour évaluer la diversité génétique (richesse allélique et hétérozygotie attendue, ([He]), déséquilibre de parenté des multiple loci ( ISA ) et structure de la population. Un degré hautement similaire de diversité génétique a été observé parmi les trois populations de parasites étudiées (He = 0,76; P > 0,05). A l'exception de Msambweni, une association aléatoire entre les microsatellites a été observée, indiquant une forte reproduction des parasites. Une structure génétique faible à modérée (FST  = 0,022-0,076; P < 0,0001) a été observée avec seulement 5% de variance dans la fréquence des allèles observée parmi les populations. Cette étude montre que la diversité génétique des populations de P. falciparum à la frontière entre le Kenya et l'Ouganda est comparable à celle des populations de parasites à l'intérieur du Kenya. De plus, la diversité génétique élevée, la panmixia et la structure démographique faible dans cette étude soulignent l'aptitude des populations de P. falciparum du Kenya à résister aux interventions de lutte contre le paludisme.

Plasmodium falciparum histidine-rich protein (PfHRP2 and 3) diversity in Western and Coastal Kenya.

Plasmodium falciparum histidine-rich proteins 2 (PfHRP2) based RDTs are advocated in falciparum malaria-endemic regions, particularly when quality microscopy is not available. However, diversity and any deletion in the pfhrp2 and pfhrp3 genes can affect the performance of PfHRP2-based RDTs. A total of 400 samples collected from uncomplicated malaria cases from Kenya were investigated for the amino acid repeat profiles in exon 2 of pfhrp2 and pfhrp3 genes. In addition, PfHRP2 levels were measured in 96 individuals with uncomplicated malaria. We observed a unique distribution pattern of amino acid repeats both in the PfHRP2 and PfHRP3. 228 PfHRP2 and 124 PfHRP3 different amino acid sequences were identified. Of this, 214 (94%) PfHRP2 and 81 (65%) PfHRP3 amino acid sequences occurred only once. Thirty-nine new PfHRP2 and 20 new PfHRP3 amino acid repeat types were identified. PfHRP2 levels were not correlated with parasitemia or the number of PfHRP2 repeat types. This study shows the variability of PfHRP2, PfHRP3 and PfHRP2 concentration among uncomplicated malaria cases. These findings will be useful to understand the performance of PfHRP2-based RDTs in Kenya.

PfHRP2-PfHRP3 diversity among Kenyan isolates and comparative evaluation of PfHRP2/pLDH malaria RDT with microscopy and nested PCR methodologies.

Rapid diagnostic tests (RDT) are valuable tools that support prudent and timely use of antimalarial drugs, particularly if reliable microscopy is not available. However, the performance and reliability of these tests vary between and within geographical regions. The present study evaluated the performance of routine malaria RDT in Kenyan febrile patients in Busia County, Kenya. A cross sectional study design was employed to recruit febrile patients attending health facilities between August and November 2016. A total of 192 febrile patients who were slide positive and negative were evaluated for their infection status by nested PCR and RDTs (PfHRP2/pLDH). In addition, P. falciparum diversity of the histidine-rich proteins 2 and 3, that influences the RDT test results were determined. All individuals were P. falciparum positive. Among the investigated 192 febrile patients, 76 (40%) were positive by microscopy, 101 (53%) by RDTs and 80 (42%) were PCR positive. The performance of the CareStart™ HRP2/pLDH (pf) RDTs was better than microscopy (Sensitivity 94%; Specificity 75%) and Nucleic acid testing (sensitivity 95%, specificity 77%) with high negative predictive values, indicating the suitability of the RDT in routine practice. Specific pfhrp2/pfhrp3 deletions shown to associate with RDT false negativity was not observed. However, high genetic diversity among pfhrp2 gene was observed. Eleven new PfHRP2 and nine PfHRP3 repeats were observed. False positivity by microscopy and under reporting of infections may thus be a barrier in malaria control and elimination programs. The HRP2/pLDH(Pf) based RDT yet demonstrate to be an effective tool for malaria surveillance program.

Minor spliceosome and disease.

The U12-dependent (minor) spliceosome excises a rare group of introns that are characterized by a highly conserved 5' splice site and branch point sequence. Several new congenital or somatic diseases have recently been associated with mutations in components of the minor spliceosome. A common theme in these diseases is the detection of elevated levels of transcripts containing U12-type introns, of which a subset is associated with other splicing defects. Here we review the present understanding of minor spliceosome diseases, particularly those associated with the specific components of the minor spliceosome. We also present a model for interpreting the molecular-level consequences of the different diseases.

The emerging role of minor intron splicing in neurological disorders.

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5' and 3' ends and are recognized by a separate pre-mRNA processing machinery denoted as the minor spliceosome. This spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on minor spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.

TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways.

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.