rRNA precursor
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Processing of rRNA in mammalian cells includes a series of cleavages of the primary 47S transcript and results in producing three rRNAs: 18S, 28S and 5.8S. The sequence of the main processing events in human cells has been established, but little is yet known about the dynamics of this process, especially the dynamics of its early stages. In the present study, we used real-time PCR to measure levels of pre-rRNA after inhibition of transcription with actinomycin D. Thus we could estimate the half-life time of rRNA transcripts in two human-derived cell lines, HeLa and LEP (human embryonic fibroblasts), as well as in mouse NIH 3T3 cells. The primary transcripts seemed to be more stable in the human than in the murine cells. Remarkably, the graphs in all cases showed more or less pronounced lag phase, which may reflect preparatory events preceding the first cleavage of the pre-rRNA. Additionally, we followed the dynamics of the decay of the 5'ETS fragment which is degraded only after the formation of 41S rRNA. According to our estimates, the corresponding three (or four) steps of the processing in human cells take five to eight minutes.
- MeSH
- buňky NIH 3T3 MeSH
- daktinomycin farmakologie MeSH
- genetická transkripce účinky léků MeSH
- HeLa buňky MeSH
- lidé MeSH
- myši MeSH
- posttranskripční úpravy RNA genetika MeSH
- prekurzory RNA * genetika metabolismus MeSH
- RNA ribozomální genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.
- MeSH
- genetická transkripce MeSH
- klíšťová encefalitida genetika metabolismus virologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- prekurzory RNA MeSH
- proteosyntéza genetika MeSH
- RNA ribozomální genetika metabolismus MeSH
- RNA-polymerasa I genetika metabolismus MeSH
- viry klíšťové encefalitidy fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.
- MeSH
- esenciální geny * MeSH
- malá jadérková RNA metabolismus MeSH
- posttranskripční úpravy RNA * MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA ribozomální 18S metabolismus MeSH
- Trypanosoma brucei brucei enzymologie metabolismus MeSH
- umlčování genů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE: Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing.
There are numerous data suggesting that two key steps in gene expression-transcription and splicing influence each other closely. For a long time it was known that chromatin modifications regulate transcription, but only recently it was shown that chromatin and histone modifications play a significant role in pre-mRNA splicing. Here we summarize interactions between splicing machinery and chromatin and discuss their potential functional significance. We focus mainly on histone acetylation and methylation and potential mechanisms of their role in splicing. It seems that whereas histone acetylation acts mainly by alterating the transcription rate, histone methylation can also influence splicing directly by recruiting various splicing components.
- MeSH
- chromatin genetika metabolismus MeSH
- genetická transkripce genetika MeSH
- lidé MeSH
- nukleoproteiny metabolismus MeSH
- prekurzory RNA genetika MeSH
- sestřih RNA MeSH
- transkripční faktory metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- biologické modely MeSH
- buněčné jádro metabolismus MeSH
- molekulární struktura MeSH
- prekurzory RNA genetika chemie metabolismus MeSH
- sestřih RNA MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Nucleolin is the major nucleolar protein of animal, plant and yeast proliferating cells. It is enriched in the most soluble nuclear or nucleolar protein extract, containing ribonucleoproteins, from which it has been purified. It has a tripartite structure in which each domain accounts for different functions. Despite its multifunctionality, the best characterized role of nucleolin is in the primary cleavage of pre-rRNA, an early step of ribosome biogenesis. In the nucleolus of proliferating cells, nucleolin is mostly located in the dense fibrillar component, following a vectorial pattern, from the periphery of fibrillar centers outwards. This pattern is lost in quiescent cells in which nucleolin is present in low levels. Nucleolin is the most phosphorylated protein of the soluble nuclear extract. It is phosphorylated by casein kinase II and CDKA, and phosphorylation is closely associated with the role of nucleolin in proliferating cells. During mitosis, nucleolin is transported from the mother to the daughter cell nucleolus in the form of processing particles, together with pre-rRNA precursors and other nucleolar proteins. It forms part of prenucleolar bodies and plays a role in nucleologenesis. Recent studies on the nucleolin function, carried out on samples with inactivated nucleolin genes (siRNA downregulated or mutants) have evidenced that nucleolin is absolutely essential for cell proliferation, for the organization of the nucleolus and for transcription and processing of pre-rRNA. In plants, nucleolin controls the auxin responsiveness, thus being involved in the regulation of plant development.
- MeSH
- biogeneze organel MeSH
- buněčný cyklus fyziologie genetika MeSH
- fosfoproteiny biosyntéza genetika chemie MeSH
- fosforylace fyziologie genetika MeSH
- jaderné proteiny biosyntéza genetika chemie MeSH
- klinické laboratorní techniky využití MeSH
- kvasinky MeSH
- kyseliny indoloctové MeSH
- prekurzory RNA biosyntéza genetika chemie MeSH
- proliferace buněk MeSH
- proteiny vázající RNA biosyntéza genetika chemie MeSH
- ribonukleoproteiny malé jadérkové biosyntéza MeSH
- ribozomy genetika chemie MeSH
- rostliny MeSH
- statistika jako téma MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH
