Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

Abstract

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.

Functional Genomic Analysis of Human Mitochondrial RNA Processing

“…The short-lived ND1 and ND2 transcripts are increased, whereas the long-lived COX1-3 transcripts are actually decreased, suggesting that SUPV3L1 may not be responsible for all degradation within the mitochondria. These contrasting effects have been previously demonstrated (Szczesny et al, 2010), but only for a subset of mt-mRNAs, and we present here data for all transcripts simultaneously. For the TRNF_RNR1 and TRNV_RNR2 junctions, which we expect to be a readout for transcriptional initiation, hairpins targeting SUPV3L1 are the second strongest inducers of expression (Figure 2,C3).…”

“…The helicase SUPV3L1, in complex with polynucleotide phosphorylase (PNPT1), has been implicated in the degradation of the light-strand transcripts, and a dominant negative form of either gene stabilizes light strand noncoding transcripts and some short-lived heavy strand mt-mRNAs (Borowski et al, 2013; Szczesny et al, 2010). However, both RNAi and dominant negative experiments targeting SUPV3L1 or PNPT1 actually decrease levels of the long-lived mt-mRNA COX1, indicating additional degradation or feedback mechanisms may exist.…”

The human Suv3 helicase interacts with replication protein A and flap endonuclease 1 in the nucleus

“…On the other hand, the stimulatory effect of hSuv3-K213A might still be sufficient to fulfil the nuclear role of hSuv3. A potential ATP-independent role of hSuv3 could explain why expression of a nuclear dominant-negative version of hSuv3 did not affect the morphology or growth rate of the cell [15]. …”

“…It appears to have a central role in mitochondrial RNA metabolism, and its knockdown is associated with accumulation of truncated mitochondrial RNA species, and decrease in mtDNA (mitochondrial DNA) copy number and mitochondrial protein expression, eventually resulting in cell death [12–14]. To date, no orthologue of the Dss1p nuclease has been found in humans, and results proposing the PNPase (polynucleotide phosphorylase) as a possible alternative [6,15] remain a matter of controversy due to the lack of PNPase in the mitochondrial matrix [16]. …”

Rarely at rest

“…In human mitochondria the partner of PNPase is likely to be DExH/D helicase hSuv3. 86,87 The crystal structure of the human helicase hSuv3p, thought to interact with human PNPase has also recently been solved and is shown in Figure 2. 88 The hSuv3 protein has a similar duplicated RecA-like core with additional strands and auxiliary α-helical domains at both termini, and the C-terminal domain helps to cradle the single stranded RNA on the surface of the RecA-like domains.…”