UHMK1 is a novel splicing regulatory kinase.

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

A Novel Intronic KMT2D Variant as a Cause of Kabuki Syndrome: A Case Report.

Kabuki syndrome (KS) is an autosomal dominant genetic disorder in which most cases are caused by de novo mutations. KS type 1 is caused by mutations in KMT2D (OMIM: #147920) and is more common. KS type 2 is caused by mutations in KDM6A (OMIM: #300867). Both genes encode proteins that modify histones and are involved in epigenetic regulation. The enzyme histone-lysine N-methyltransferase 2D, the product of KMT2D, is expressed in most adult tissues and is essential for early embryonic development. The main clinical manifestations of KS include dysmorphic facial features, such as elongated palpebral fissures, eversion of the lateral third of the lower eyelids, and short nasal columella with a broad and depressed nasal tip. Additionally, patients also present with skeletal abnormalities, dermatoglyphic features, mild-to-moderate intellectual disability, hearing loss, and postnatal growth deficiency. We describe an 11-year-old girl from Colombia, who presented with characteristic clinical signs of KS. Genetic studies showed a KMT2D intronic variant (KMT2D NM_003482.3: c.511-2A> T) as a cause of KS.

Human RNF113A participates of pre-mRNA splicing in vitro.

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Inhibición parcial de dos genes que codifican para proteínas del empalmosoma en Giardia intestinalis / Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.

Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis.

INTRODUCTION: Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Plaquetas: papéis tradicionais e não tradicionais na hemostasia, na inflamação e no câncer / Platelets: traditional and nontraditional roles in hemostasis, inflammation and cancer

A principal e mais conhecida função plaquetária ainda está relacionada à parada de sangramento após um dano vascular. No entanto, plaquetas estão envolvidas em diversos processos, tais como iniciar e amplificar a inflamação, interagir com células da resposta imune, além de participar na progressão tumoral, angiogênese e metástase. Neste sentido, está claro que plaquetas apresentam funções no processo inflamatório e podem influenciar respostas imune, além de desordens plaquetárias autoimune erelacionadas a presença de auto-anticorpos após transfusões, comopor exemplo, na lesão pulmonar aguda associada à transfusão. Após muita especulação, recentes observações têm estabelecido novos paradigmas relacionando plaquetas à biologia molecular. Plaquetas humanas contêm fatores de spliceossomo, incluindo pequenos RNAs nucleares, proteínas de splicing e pre-mRNA endógenos. Outro ponto importante é o controle do número de plaquetas circulantes, resultado do equilíbrio entre a produção e destruição dessas células. Assim, é proposto um processo de morte programada da célula anucleada que determina seu tempo de vida. Esse processo é alvo de especulações desde a década de 60 e ainda permanece em discussão. A noção geral de que plaquetas funcionais são importantes para o sucesso de processos hematogênicos corroboram com inovações experimentais e também ligam a processos de interação plaquetas-células tumorais e seu microambiente que regula a progressão maligna. Plaquetas contribuem na sobrevivência e disseminação de células tumorais. Desta forma, discutimos aqui os mecanismos pelos quais as plaquetas atuam na imunidade, na inflamação e no câncer, uma vez que estas pequenas células são mais versáteis do que se pensava.

The principal and the most known function of platelets still remains stopping hemorrhage following vascular injury. However, platelets are involved in diverse processes such as triggering inflammation, participating in the immune response, besides tumor progression, angiogenesis, and metastasis. In this sense, it is becoming increasingly clear that platelets display inflammatory functions and can influence both innate and adaptive immune responses, such as autoimmune and alloimmune platelet disorders, and transfusion-related acute lung injury (TRALI). Despite much speculation recent observations have established new paradigms relevant to influence of platelets on molecular biology. Primary human platelets contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. Other point is, like all lineages of blood cells, the steady state number of mature platelets is the result of a balance between their production and destruction. Thus, it isproposed a programmed anuclear cell death delimits platelet lifespan is subject of speculation since the 1960s and has remainedelusive. The general notion that functional platelets are importantfor successful hematogenous tumor metastasis dates more than 4 decades and has been corroborated in numerous experimentalsettings. The dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator ofmalignant progression. These contributions of platelets to tumorcell survival and spread suggest platelets as a new avenue forresearch. Here, we discuss the mechanisms by which plateletscontribute to immunity, inflammation, and cancer, since thesesmall cells are more versatile than we once thought.