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1.
Genet Med ; 26(2): 101023, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37947183

RESUMO

PURPOSE: We sought to delineate a multisystem disorder caused by recessive cysteine-rich with epidermal growth factor-like domains 1 (CRELD1) gene variants. METHODS: The impact of CRELD1 variants was characterized through an international collaboration utilizing next-generation DNA sequencing, gene knockdown, and protein overexpression in Xenopus tropicalis, and in vitro analysis of patient immune cells. RESULTS: Biallelic variants in CRELD1 were found in 18 participants from 14 families. Affected individuals displayed an array of phenotypes involving developmental delay, early-onset epilepsy, and hypotonia, with about half demonstrating cardiac arrhythmias and some experiencing recurrent infections. Most harbored a frameshift in trans with a missense allele, with 1 recurrent variant, p.(Cys192Tyr), identified in 10 families. X tropicalis tadpoles with creld1 knockdown displayed developmental defects along with increased susceptibility to induced seizures compared with controls. Additionally, human CRELD1 harboring missense variants from affected individuals had reduced protein function, indicated by a diminished ability to induce craniofacial defects when overexpressed in X tropicalis. Finally, baseline analyses of peripheral blood mononuclear cells showed similar proportions of immune cell subtypes in patients compared with healthy donors. CONCLUSION: This patient cohort, combined with experimental data, provide evidence of a multisystem clinical syndrome mediated by recessive variants in CRELD1.


Assuntos
Transtornos do Neurodesenvolvimento , Reinfecção , Humanos , Leucócitos Mononucleares , Síndrome , Fenótipo , Arritmias Cardíacas/genética , Transtornos do Neurodesenvolvimento/genética , Moléculas de Adesão Celular/genética , Proteínas da Matriz Extracelular/genética
3.
Genes Dev ; 29(18): 1891-6, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26338418

RESUMO

We show that a common polymorphic variant in the ERCC5 5' untranslated region (UTR) generates an upstream ORF (uORF) that affects both the background expression of this protein and its ability to be synthesized following exposure to agents that cause bulky adduct DNA damage. Individuals that harbor uORF1 have a marked resistance to platinum-based agents, illustrated by the significantly reduced progression-free survival of pediatric ependymoma patients treated with such compounds. Importantly, inhibition of DNA-PKcs restores sensitivity to platinum-based compounds by preventing uORF1-dependent ERCC5 expression. Our data support a model in which a heritable 5' noncoding mRNA element influences individuals' responses to platinum-based chemotherapy.


Assuntos
Regiões 5' não Traduzidas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Endonucleases/genética , Endonucleases/metabolismo , Ependimoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta/genética , Polimorfismo Genético/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA , Ependimoma/tratamento farmacológico , Ependimoma/mortalidade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos
4.
Biochem Soc Trans ; 38(6): 1581-6, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21118130

RESUMO

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.


Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Transativadores/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Conformação de Ácido Nucleico , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética
5.
Nucleic Acids Res ; 37(17): 5881-93, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19654240

RESUMO

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.


Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , Receptor de Insulina/genética , Animais , Sequência de Bases , Linhagem Celular , Humanos , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , RNA Mensageiro/química , Ratos
6.
Mol Cell Biol ; 29(6): 1565-74, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19124605

RESUMO

Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5' end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5' untranslated region (5'-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5'-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribossomos/fisiologia , Regiões 5' não Traduzidas , Códon de Iniciação , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Células HeLa , Humanos , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
7.
Mol Cell Biol ; 28(1): 40-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17967896

RESUMO

The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.


Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribossomos/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB , Polirribossomos/metabolismo , Ligação Proteica , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/classificação , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/genética
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