RESUMO
Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Poliadenilação/genética , Isoformas de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/fisiologia , Animais , Estruturas do Núcleo Celular/ultraestrutura , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Células NIH 3T3 , Proteínas Nucleares/isolamento & purificação , Poliadenilação/fisiologia , Estrutura Terciária de Proteína , Isoformas de RNA/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Recently, we cloned and began to characterize a new N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca(2+) permeability, decrease in Mg(2+) sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8 (P8) and similarly found to be relatively insensitive to Mg(2+) block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.
