RESUMO
In eukaryotic cells, the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs is mediated by an assemblyosome, the survival of motor neurons (SMN) complex. The SMN complex, composed of SMN and the Gemins (2-7), binds to the Sm proteins and to snRNAs and constructs the heptameric rings, the common cores of Sm proteins, on the Sm site (AU(56)G) of the snRNAs. We have determined the specific sequence and structural features of snRNAs for binding to the SMN complex and Sm core assembly. The minimal SMN complex-binding domain in snRNAs (except U1) is composed of an Sm site and a closely adjacent 3'stem-loop. Remarkably, the specific sequence of the stemloop is not important for SMN complex binding, but it must be located within a short distance of the 3'end of the RNA for an Sm core to assemble. This minimal snRNA-defining "snRNP code" is recognized by the SMN complex, which binds to it directly and with high affinity and assembles the Sm core. The recognition of the snRNAs is provided by Gemin5, a component of the SMN complex that directly binds the snRNP code. Gemin5 is a novel RNA-binding protein that is critical for snRNP biogenesis. Thus, the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells. The function of the SMN complex, previously unanticipated because RNP biogenesis was believed to occur by self-assembly, confers stringent specificity on otherwise potentially illicit RNA-protein interactions.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Sequência de Bases , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/biossíntese , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Spliceossomos/metabolismoRESUMO
Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)(+) mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.
Assuntos
Proteínas de Transporte/metabolismo , Endorribonucleases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Proteína p120 Ativadora de GTPase/metabolismo , Regiões 3' não Traduzidas/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA Helicases , Endorribonucleases/genética , Fibroblastos/citologia , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Serina/genética , Serina/metabolismo , Especificidade por Substrato , Proteína p120 Ativadora de GTPase/genéticaRESUMO
Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.
