ABSTRACT
Accurate tRNA processing is crucial for human mitochondrial genome expression, but the mechanisms of mt-tRNA cleavage and the key enzymes involved in this process are poorly characterized. At least two activities are required for proper mt-tRNA maturation: RNase P cleaving precursor molecules at the 5' end and tRNase Z at the 3' end. In human mitochondria only RNase P has been identified so far. Using RT-PCR and northern blot analyses we found that silencing of the human ELAC2 gene results in impaired 3' end of mt-tRNAs. We demonstrate this for several mitochondrial tRNAs, encoded on both mtDNA strands, including tRNA (Val) , tRNA (Lys) , tRNA (Arg) , tRNA (Gly) , tRNA (Leu(UUR)) and tRNA (Glu) . The silencing of the MRPP1 gene that encodes a subunit of mtRNase P resulted in inhibition of both 5' and 3' processing. We also demonstrate the double mitochondrial/nuclear localization of the ELAC2 protein using immunofluorescence. Our results indicate that ELAC2 functions as a tRNase Z in human mitochondria and suggest that mt-tRNase Z preferentially cleaves molecules already processed by the proteinaceous mtRNase P.
Subject(s)
Mitochondria/genetics , Neoplasm Proteins/metabolism , RNA 3' End Processing/genetics , RNA, Transfer/genetics , RNA/metabolism , Cell Line, Tumor , Cell Nucleus/enzymology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Endoribonucleases/metabolism , HEK293 Cells , HeLa Cells , Humans , Mitochondria/enzymology , Mitochondria/metabolism , Neoplasm Proteins/genetics , RNA/genetics , RNA Interference , RNA, Mitochondrial , RNA, Small Interfering , RNA, Transfer/metabolism , Ribonuclease P/metabolismABSTRACT
Protein complexes responsible for RNA degradation play important role in three key aspects of RNA metabolism: they control stability of physiologically functional transcripts, remove the unnecessary RNA processing intermediates and destroy aberrantly formed RNAs. In mitochondria the post-transcriptional events seem to play a major role in regulation of gene expression, therefore RNA turnover is of particular importance. Despite many years of research, the details of this process are still a challenge. This review summarizes emerging landscape of interplay between the Suv3p helicase (SUPV3L1, Suv3), poly(A) polymerase and polynucleotide phosphorylase in controlling RNA degradation in human mitochondria.
Subject(s)
Mitochondria/metabolism , RNA/metabolism , DEAD-box RNA Helicases/metabolism , Humans , In Vitro Techniques , Mitochondria/genetics , Models, Biological , Poly U/metabolism , Polyadenylation , Polynucleotide Adenylyltransferase/metabolism , Polyribonucleotide Nucleotidyltransferase/metabolism , RNA/genetics , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Mitochondrial , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3'-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.
