RESUMEN

Heart failure (HF) is associated with global changes in gene expression. Alternative mRNA splicing (AS) is a key regulatory mechanism underlying these changes. However, the whole status of molecules involved in the splicing process in human HF is unknown. Therefore, we analysed the spliceosome transcriptome in cardiac tissue (n = 36) from control subjects and HF patients (with ischaemic (ICM) and dilated (DCM) cardiomyopathies) using RNA-seq. We found greater deregulation of spliceosome machinery in ICM. Specifically, we showed widespread upregulation of the E and C complex components, highlighting an increase in SNRPD2 (FC = 1.35, p < 0.05) and DHX35 (FC = 1.34, p < 0.001) mRNA levels. In contrast, we observed generalised downregulation of the A complex and cardiac-specific AS factors, such as the multifunctional protein PCBP2 (FC = -1.29, p < 0.001) and the RNA binding proteins QKI (FC = -1.35, p < 0.01). In addition, we found a relationship between SNPRD2 (an E complex component) and the left ventricular mass index in ICM patients (r = 0.779; p < 0.01). On the other hand, we observed the specific underexpression of DDX46 (FC = -1.29), RBM17 (FC = -1.33), SDE2 (FC = -1.35) and RBFOX1 (FC = -1.33), p < 0.05, in DCM patients. Therefore, these aetiology-related alterations may indicate the differential involvement of the splicing process in the development of ICM and DCM.

Asunto(s)

Empalme Alternativo , Insuficiencia Cardíaca , Factores de Empalme de ARN , Empalmosomas , Transcriptoma , Humanos , Empalmosomas/metabolismo , Empalmosomas/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Anciano , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Miocardio/metabolismo , Miocardio/patología , Perfilación de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

Asunto(s)

Discapacidades del Desarrollo , ARN , Enfermedades Raras , Humanos , Discapacidades del Desarrollo/genética , Enfermedades Raras/genética , ARN/genética , Empalme del ARN/genética , Empalmosomas/genética

RESUMEN

INTRODUCTION: Allogeneic Hematopoietic cell transplantation (allo-HCT) remains the only curative therapy for myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). The impact of spliceosome mutations on allo-HCT outcome is unclear and further understanding is needed to assess the implications of this class of mutations on risk of relapse, overall survival (OS) and non-relapse mortality (NRM) in order to make decision regarding timing of allo-HCT. We examined the allo-HCT outcomes of MDS/CMML patients based on their spliceosome mutation profile to understand the impact of these mutations on transplant outcomes. OBJECTIVE: To compare outcomes of MDS/CMML patients with and without spliceosome mutations undergoing allo-HCT. METHODS: This is a single institution, retrospective study of MDS/CMML patients who underwent allo-HCT with myeloablative or reduced intensity conditioning (RIC) regimen at City of Hope from January 2016 to December 2021. Among them, patients who underwent molecular mutation profiling by NGS (Next Generation Sequencing) for a set of genes known to be mutated in myeloid neoplasms are included in this analysis. We compared OS, relapse free survival, NRM and acute/chronic graft versus host disease (GVHD) incidence between the spliceosome-mutated and unmutated groups. RESULTS: We identified 258 consecutive MDS/CMML patients who underwent allo-HCT. Of these, 126 (48.8 %) patients had molecular profiling done among whom 57 (45.2 %) patients carried a spliceosome mutation. 84.9 % of patients had MDS and 55.6 % underwent a matched unrelated donor transplant. The median age for the whole cohort was 66 years (range 12-77).78.6 % and 73.7 % received RIC in the spliceosome and non-spliceosome groups, respectively. The 2-year OS for the whole cohort was 66.5 % (95 %CI 0.55-0.75) with a day 100 NRM of 7.1 % and 2-year cumulative incidence of relapse of 20 %. Grade II-IV acute GVHD at day 100 was 36.3 % (95 % CI 0.27-0.44) and any chronic GVHD at 2-years was 48.4 % (95 % CI 0.37-0.58). Patients who carried a spliceosome mutation had a significantly better 2-year survival of 83.8 % vs 55.9 % in the non-spliceosome group (P=0.002) and a better PFS of 73.7 % vs 50.0 % (P=0.007). There was no difference in the cumulative incidence of relapse at 2-years 15.9 % vs 18.5 % (P=0.59) between two groups but the spliceosome group had a significantly lower NRM at 2-years 10.4 % vs 31.5 % (P=0.009). There was no difference in incidence of acute or chronic GVHD between the two groups. CONCLUSIONS: Among patients with MDS or CMML who underwent allo-HCT, our study shows better OS for patients who have spliceosome mutations due to lower NRM compared to those carrying non- spliceosome mutations. This favorable outcome of the spliceosome-mutated patients could have implications for timing of allo-HCT, particularly for patients in the intermediate MDS prognostic risk groups.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Leucemia Mielomonocítica Crónica , Mutación , Síndromes Mielodisplásicos , Empalmosomas , Trasplante Homólogo , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/terapia , Síndromes Mielodisplásicos/mortalidad , Empalmosomas/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Masculino , Femenino , Persona de Mediana Edad , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/terapia , Leucemia Mielomonocítica Crónica/mortalidad , Estudios Retrospectivos , Adulto , Anciano , Acondicionamiento Pretrasplante/métodos , Tasa de Supervivencia , Pronóstico , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/genética , Adulto Joven

RESUMEN

The two transesterification reactions of pre-mRNA splicing require highly complex yet well-controlled rearrangements of small nuclear RNAs and proteins (snRNP) in the spliceosome. The efficiency and accuracy of these reactions are critical for gene expression, as almost all human genes pass through pre-mRNA splicing. Key parameters that determine the splicing outcome are the length of the intron, the strengths of its splicing signals and gaps between them, and the presence of splicing controlling elements. In particular, the gap between the branchpoint (BP) and the 3' splice site (ss) of introns is a major determinant of the splicing efficiency. This distance falls within a small range across the introns of an organism. The constraints exist possibly because BP and 3'ss are recognized by BP-binding proteins, U2 snRNP and U2 accessory factors (U2AF) in a coordinated manner. Furthermore, varying distances between the two signals may also affect the second transesterification reaction since the intervening RNA needs to be accurately positioned within the complex RNP machinery. Splicing such pre-mRNAs requires cis-acting elements in the RNA and many trans-acting splicing regulators. Regulated pre-mRNA splicing with BP-distant 3'ss adds another layer of control to gene expression and promotes alternative splicing.

Asunto(s)

Intrones , Sitios de Empalme de ARN , Empalme del ARN , Humanos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalmosomas/metabolismo , Empalmosomas/genética , Animales

RESUMEN

The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.

Asunto(s)

Proteínas Reguladoras de la Apoptosis , Proteínas de Ciclo Celular , Proteína del Grupo de Complementación A de la Anemia de Fanconi , Anemia de Fanconi , Empalme del ARN , Factor de Empalme U2AF , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Reparación del ADN , Endodesoxirribonucleasas , Exones , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Unión Proteica , Precursores del ARN/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Empalmosomas/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/metabolismo , Factor de Empalme U2AF/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo

RESUMEN

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.

Asunto(s)

Núcleo Celular , Empalme del ARN , Proteínas de Unión al ARN , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Precursores del ARN/metabolismo , Precursores del ARN/genética , Mutación , Empalmosomas/metabolismo , Empalmosomas/genética , Células HeLa , Células HEK293

RESUMEN

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Asunto(s)

Encéfalo , Trastornos del Neurodesarrollo , ARN Nuclear Pequeño , Humanos , ARN Nuclear Pequeño/genética , Trastornos del Neurodesarrollo/genética , Femenino , Masculino , Encéfalo/metabolismo , Heterocigoto , Alelos , Síndrome , Empalmosomas/genética , Animales

RESUMEN

Pre-mRNA splicing, the removal of introns and ligation of flanking exons, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins, assembles on introns via a complex pathway before catalyzing the two transesterification reactions necessary for splicing. All of these steps have the potential to be highly regulated to ensure correct mRNA isoform production for proper cellular function. While Saccharomyces cerevisiae (yeast) has a limited set of intron-containing genes, many of these genes are highly expressed, resulting in a large number of transcripts in a cell being spliced. As a result, splicing regulation is of critical importance for yeast. Just as in humans, yeast splicing can be influenced by protein components of the splicing machinery, structures and properties of the pre-mRNA itself, or by the action of trans-acting factors. It is likely that further analysis of the mechanisms and pathways of splicing regulation in yeast can reveal general principles applicable to other eukaryotes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Asunto(s)

Precursores del ARN , Empalme del ARN , Saccharomyces cerevisiae , Empalmosomas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Empalmosomas/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Inmunidad de la Planta , Precursores del ARN , Empalme del ARN , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalmosomas/metabolismo , Empalmosomas/genética

RESUMEN

At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

Asunto(s)

Candida albicans , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Empalmosomas , Candida albicans/genética , Candida albicans/patogenicidad , Candida albicans/crecimiento & desarrollo , Candida albicans/fisiología , Candida albicans/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Morfogénesis/genética , Empalme del ARN , Virulencia , Hifa/crecimiento & desarrollo , Hifa/genética , Intrones/genética

RESUMEN

Classification of introns, which is crucial to understanding their evolution and splicing, has historically been binary and has resulted in the naming of major and minor introns that are spliced by their namesake spliceosome. However, a broad range of intron consensus sequences exist, leading us to here reclassify introns as minor, minor-like, hybrid, major-like, major and non-canonical introns in 263 species across six eukaryotic supergroups. Through intron orthology analysis, we discovered that minor-like introns are a transitory node for intron conversion across evolution. Despite close resemblance of their consensus sequences to minor introns, these introns possess an AG dinucleotide at the -1 and -2 position of the 5' splice site, a salient feature of major introns. Through combined analysis of CoLa-seq, CLIP-seq for major and minor spliceosome components, and RNAseq from samples in which the minor spliceosome is inhibited we found that minor-like introns are also an intermediate class from a splicing mechanism perspective. Importantly, this analysis has provided insight into the sequence elements that have evolved to make minor-like introns amenable to recognition by both minor and major spliceosome components. We hope that this revised intron classification provides a new framework to study intron evolution and splicing.

Asunto(s)

Evolución Molecular , Intrones , Empalme del ARN , Empalmosomas , Intrones/genética , Empalmosomas/genética , Humanos , Sitios de Empalme de ARN , Animales , Secuencia de Consenso , Eucariontes/genética , Eucariontes/clasificación , Secuencia de Bases

RESUMEN

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.

Asunto(s)

Histona Desacetilasas , Histonas , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Histonas/metabolismo , Histonas/genética , Humanos , Animales , Empalmosomas/metabolismo , Empalmosomas/genética , Acetilación , Drosophila/genética , Drosophila/metabolismo , Transcripción Genética/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas Represoras

RESUMEN

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Asunto(s)

Factores de Empalme de ARN , Empalme del ARN , ARN Nuclear Pequeño , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Empalmosomas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Empalmosomas/genética , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/química , Intrones/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Microscopía por Crioelectrón , Mutación , Unión Proteica , Dominio Catalítico , Alelos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/química , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U5 , ARN Helicasas

RESUMEN

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

Asunto(s)

Axonema , Intrones , Proteínas Protozoarias , Empalme del ARN , Proteínas de Unión al ARN , Intrones/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Animales , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Masculino , Axonema/metabolismo , Femenino , Gametogénesis/genética , Empalmosomas/metabolismo , Empalmosomas/genética , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/metabolismo , Malaria/parasitología , Plasmodium/genética , Plasmodium/metabolismo

RESUMEN

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes.

Asunto(s)

Intrones , Intrones/genética , Animales , Humanos , Arabidopsis/genética , Empalmosomas/genética , Empalmosomas/metabolismo

RESUMEN

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of ß-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 ß-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

Asunto(s)

Intrones , Globinas beta , Humanos , Globinas beta/genética , Empalme del ARN , Talasemia beta/genética , Talasemia beta/diagnóstico , Sitios de Empalme de ARN , Eliminación de Secuencia , Empalmosomas/genética , Empalmosomas/metabolismo

RESUMEN

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.

Asunto(s)

Proteínas Argonautas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ARN Nuclear Pequeño , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Animales , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Empalme Alternativo/genética , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Empalmosomas/metabolismo , Empalmosomas/genética

RESUMEN

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that the conserved U6 snRNA m6A methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of METT-10 in C. elegans and METTL16 in humans primarily leads to alternative splicing at 5' splice sites with an adenosine at +4 position. In addition, METT-10 is required for splicing of weak 3' cis- and trans-splice sites. We identified a significant overlap between METT-10 and the conserved splicing factor SNRNP27K in regulating 5' splice sites with +4A. Finally, we show that editing endogenous 5' splice site +4A positions to +4U restores splicing to wild-type positions in a mett-10 mutant background, supporting a direct role for U6 snRNA m6A modification in 5' splice site recognition. We conclude that the U6 snRNA m6A modification is important for accurate and efficient pre-mRNA splicing.

Asunto(s)

Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Metiltransferasas , Precursores del ARN , Sitios de Empalme de ARN , Empalme del ARN , ARN Nuclear Pequeño , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Animales , Precursores del ARN/metabolismo , Precursores del ARN/genética , Humanos , Metiltransferasas/metabolismo , Metiltransferasas/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Empalme Alternativo , Empalmosomas/metabolismo , Empalmosomas/genética

RESUMEN

Most people with intellectual disability (ID) do not receive a molecular diagnosis following genetic testing. To identify new etiologies of ID, we performed a genetic association analysis comparing the burden of rare variants in 41,132 noncoding genes between 5,529 unrelated cases and 46,401 unrelated controls. RNU4-2, which encodes U4 small nuclear RNA, a critical component of the spliceosome, was the most strongly associated gene. We implicated de novo variants among 47 cases in two regions of RNU4-2 in the etiology of a syndrome characterized by ID, microcephaly, short stature, hypotonia, seizures and motor delay. We replicated this finding in three collections, bringing the number of unrelated cases to 73. Analysis of national genomic diagnostic data showed RNU4-2 to be a more common etiological gene for neurodevelopmental abnormality than any previously reported autosomal gene. Our findings add to the growing evidence of spliceosome dysfunction in the etiologies of neurological disorders.

Asunto(s)

Discapacidad Intelectual , Mutación , Trastornos del Neurodesarrollo , ARN Nuclear Pequeño , Humanos , ARN Nuclear Pequeño/genética , Trastornos del Neurodesarrollo/genética , Discapacidad Intelectual/genética , Mutación/genética , Femenino , Masculino , Empalmosomas/genética , Microcefalia/genética , Microcefalia/epidemiología , Estudios de Asociación Genética , Niño

RESUMEN

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Asunto(s)

Exones , Intrones , Precursores del ARN , Sitios de Empalme de ARN , Empalme del ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , Animales , Intrones/genética , Exones/genética , Genes de Plantas , Modelos Genéticos , Empalmosomas/metabolismo , Empalmosomas/genética , Plantas/genética , Humanos , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo