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

Splicing is a critical processing step during pre-mRNA maturation in eukaryotes. The correct selection of splice sites during the early steps of spliceosome assembly is highly important and crucial for the regulation of alternative splicing. Splice site recognition and alternative splicing depend on cis-regulatory sequence elements in the RNA and trans-acting splicing factors that recognize these elements and crosstalk with the canonical splicing machinery. Structural mechanisms involving early spliceosome complexes are governed by dynamic RNA structures, protein-RNA interactions and conformational flexibility of multidomain RNA binding proteins. Here, we highlight structural studies and integrative structural biology approaches, which provide complementary information from cryo-EM, NMR, small angle scattering, and X-ray crystallography to elucidate mechanisms in the regulation of early spliceosome assembly and quality control, highlighting the role of conformational dynamics.

Asunto(s)

Empalmosomas , Empalmosomas/metabolismo , Empalmosomas/química , Humanos , Empalme del ARN , Unión Proteica , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Modelos Moleculares

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

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

Asunto(s)

Microscopía por Crioelectrón , Precursores del ARN , Empalme del ARN , ARN Nuclear Pequeño , Empalmosomas , Empalmosomas/metabolismo , Humanos , Precursores del ARN/metabolismo , Precursores del ARN/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Modelos Moleculares , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Dominio Catalítico

RESUMEN

The early-life organ development and maturation shape the fundamental blueprint for later-life phenotype. However, a multi-organ proteome atlas from infancy to adulthood is currently not available. Herein, we present a comprehensive proteomic analysis of ten mouse organs (brain, heart, lung, liver, kidney, spleen, stomach, intestine, muscle and skin) at three crucial developmental stages (1-, 4- and 8-weeks after birth) acquired using data-independent acquisition mass spectrometry. We detect and quantify 11,533 protein groups across the ten organs and obtain 115 age-related differentially expressed protein groups that are co-expressed in all organs from infancy to adulthood. We find that spliceosome proteins prevalently play crucial regulatory roles in the early-life development of multiple organs, and detect organ-specific expression patterns and sexual dimorphism. This multi-organ proteome atlas provides a fundamental resource for understanding the molecular mechanisms underlying early-life organ development and maturation.

Asunto(s)

Proteoma , Proteómica , Animales , Proteoma/metabolismo , Ratones , Femenino , Masculino , Proteómica/métodos , Riñón/metabolismo , Riñón/crecimiento & desarrollo , Empalmosomas/metabolismo , Especificidad de Órganos , Ratones Endogámicos C57BL , Encéfalo/metabolismo , Encéfalo/crecimiento & desarrollo , Hígado/metabolismo , Pulmón/metabolismo , Pulmón/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Caracteres Sexuales , Bazo/metabolismo , Bazo/crecimiento & desarrollo

Asunto(s)

ARN Helicasas DEAD-box , Factores de Empalme Serina-Arginina , Empalmosomas , Humanos , Empalmosomas/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Unión Proteica

Asunto(s)

Empalme del ARN , Empalmosomas , Animales , Humanos , Empalmosomas/química , Empalmosomas/metabolismo

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

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to ß-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Asunto(s)

Ribonucleoproteína Nuclear Pequeña U1 , Factores de Empalme Serina-Arginina , Empalmosomas , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/química , Factores de Empalme Serina-Arginina/genética , Fosforilación , Empalmosomas/metabolismo , Empalmosomas/química , Humanos , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/genética , Empalme del ARN , Unión Proteica , Precursores del ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/química

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

U6 snRNA is one of the uridine-rich non-coding RNAs, abundant and stable in various cells, function as core particles in the intron-lariat spliceosome (ILS) complex. The Increased Level of Polyploidy1-1D (ILP1) and NTC-related protein 1 (NTR1), two conserved disassembly factors of the ILS complex, facilitates the disintegration of the ILS complex after completing intron splicing. The functional impairment of ILP1 and NTR1 lead to increased U6 levels, while other snRNAs comprising the ILS complex remained unaffected. We revealed that ILP1 and NTR1 had no impact on the transcription, 3' end phosphate structure or oligo(U) tail of U6 snRNA. Moreover, we uncovered that the mutation of ILP1 and NTR1 resulted in the accumulation of ILS complexes, impeding the dissociation of U6 from splicing factors, leading to an extended half-life of U6 and ultimately causing an elevation in U6 snRNA levels. Our findings broaden the understanding of the functions of ILS disassembly factors ILP1 and NTR1, and providing insights into the dynamic disassembly between U6 and ILS.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , ARN Nuclear Pequeño , Empalmosomas , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Empalmosomas/metabolismo , Empalme del ARN , ARN de Planta/metabolismo , ARN de Planta/genética , Estabilidad del ARN/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

Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodelling for catalysis2-6, but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.

Asunto(s)

Caenorhabditis elegans , Empalmosomas , Animales , Humanos , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Microscopía por Crioelectrón , Intrones/genética , Modelos Moleculares , ARN Helicasas/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/química , Empalmosomas/metabolismo , Empalmosomas/ultraestructura , Empalmosomas/química , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo

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