ABSTRACT
Noncoding RNAs have recently been identified as essential components of the nuclear suborganelles called paraspeckles. This finding will facilitate our understanding of the molecular dynamics and physiological role of these enigmatic macromolecular structures.
Subject(s)
Cell Cycle , Cell Nucleus/metabolism , Nuclear Proteins/metabolism , RNA, Untranslated/metabolism , Animals , Humans , Models, Biological , Proto-Oncogene Proteins/genetics , RNA, Untranslated/geneticsABSTRACT
Recent transcriptome analyses have shown that thousands of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. Although the number of functionally annotated ncRNAs is still limited, they are known to be frequently retained in the nucleus, where they coordinate regulatory networks of gene expression. Some subnuclear organelles or nuclear bodies include RNA species whose identity and structural roles are largely unknown. We identified 2 abundant overlapping ncRNAs, MENepsilon and MENbeta (MENepsilon/beta), which are transcribed from the corresponding site in the multiple endocrine neoplasia (MEN) I locus and which localize to nuclear paraspeckles. This finding raises the intriguing possibility that MENepsilon/beta are involved in paraspeckle organization, because paraspeckles are, reportedly, RNase-sensitive structures. Successful removal of MENepsilon/beta by a refined knockdown method resulted in paraspeckle disintegration. Furthermore, the reassembly of paraspeckles disassembled by transcriptional arrest appeared to be unsuccessful in the absence of MENepsilon/beta. RNA interference and immunoprecipitation further revealed that the paraspeckle proteins p54/nrb and PSF selectively associate with and stabilize the longer MENbeta, thereby contributing to the organization of the paraspeckle structure. The paraspeckle protein PSP1 is not directly involved in either MENepsilon/beta stabilization or paraspeckle organization. We postulate a model for nuclear paraspeckle body organization where specific ncRNAs and RNA-binding proteins cooperate to maintain and, presumably, establish the structure.
Subject(s)
Cell Nucleus/metabolism , Proto-Oncogene Proteins/genetics , RNA, Untranslated , Dactinomycin/pharmacology , Gene Knockdown Techniques , HeLa Cells , Humans , Immunoprecipitation , Oligonucleotides, Antisense/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Pre-mRNA splicing specifically deposits the exon junction complex (EJC) onto spliced mRNA, which is important for downstream events. Here, we show that EJC components are primarily recruited to the spliceosome by association with the intron via the intron-binding protein, IBP160. This initial association of EJC components occurs in the absence of the final EJC-binding site on the exon. RNA interference (RNAi) knockdown of IBP160 arrested EJC association with cytoplasmic RNAs following nonsense-mediated decay. We propose that the intron has a crucial role in the early steps of EJC formation and is indispensable for the subsequent formation of a functional EJC.
Subject(s)
Exons , Introns , RNA Splicing , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , In Vitro Techniques , Models, Biological , RNA Interference , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Small Interfering/genetics , Spliceosomes/genetics , Spliceosomes/metabolismABSTRACT
In the human HOXA locus a number of ncRNAs are transcribed from the intergenic regions in the opposite direction to HOXA mRNAs. We observed that the genomic organization of genes for the ncRNAs and HOXA proteins is highly conserved between human and mouse. We examined the expression profiles of these ncRNAs and HOXA mRNAs in various human tissues. The expression patterns of ncRNAs in human tissues coincide with those of the adjacent HOXA mRNAs that are collinearly expressed along the anteroposterior axis. This coordinated expression was observed even in transformed tumors and cancer cell lines, suggesting that the expression of ncRNAs is prerequisite for the regulated expression of HOXA genes. HIT18844 ncRNA transcribed from the most upstream position of the HOXA cluster possesses an ultra-conserved short stretch which potentially forms an evolutionarily conserved secondary structure. Our data suggest a critical role for ncRNAs in the regulation of HOXA gene expression.
Subject(s)
Gene Expression Profiling , Homeodomain Proteins/genetics , RNA, Messenger/genetics , RNA, Untranslated/genetics , Animals , Base Sequence , Cell Line , Cell Line, Tumor , Female , HL-60 Cells , HeLa Cells , Humans , Jurkat Cells , K562 Cells , Mice , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Untranslated/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
We have examined the expression profile of selected non-coding RNAs (ncRNAs) in 11 human tissues. Among 5489 full-length cDNA clones annotated as non-protein-coding transcripts in the H-Invitational database, we chose 150 clones for further analysis based on their gene structure and EST information. Expression profiling using quantitative RT-PCR and Northern blot hybridization revealed that the majority of the selected ncRNAs exhibited tissue specificity: 67% are predominantly expressed in a restricted subset of tissues. The absolute quantification of representative ncRNAs revealed that the majority of ncRNAs are expressed as low abundance transcripts. A comparative genomic analysis revealed that only 27% of the selected ncRNAs have mouse counterparts. Since the expression patterns of the human ncRNAs having no mouse counterparts remain to be similar to those of the mouse ncRNAs, the expression patterns of the selected ncRNAs may be conserved between human and mouse.
