Reference Gene Validation via RT-qPCR for Human iPSC-Derived Neural Stem Cells and Neural Progenitors.

Correct selection of the reference gene(s) is the most important step in gene expression analysis. The aims of this study were to identify and evaluate the panel of possible reference genes in neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP) obtained from human-induced pluripotent stem cells (hiPSC). The stability of expression of genes commonly used as the reference in cells during neural differentiation is variable and does not meet the criteria for reference genes. In the present work, we evaluated the stability of expression of 16 candidate reference genes using the four most popular algorithms: the ΔCt method, BestKeeper, geNorm and NormFinder. All data were analysed using the online tool RefFinder to obtain a comprehensive ranking. Our results indicate that NormFinder is the best tool for reference gene selection in early stages of hiPSC neural differentiation. None of the 16 tested genes is suitable as reference gene for all three stages of development. We recommend using different genes (panel of genes) to normalise RT-qPCR data for each of the neural differentiation stages.

Controlling the mitochondrial antisense - role of the SUV3-PNPase complex and its co-factor GRSF1 in mitochondrial RNA surveillance.

Transcription of the human mitochondrial genome produces a vast amount of non-coding antisense RNAs. These RNA species can form G-quadraplexes (G4), which affect their decay. We found that the mitochondrial degradosome, a complex of RNA helicase SUPV3L1 (best known as SUV3) and the ribonuclease PNPT1 (also known as PNPase), together with G4-melting protein GRSF1, is a key player in restricting antisense mtRNAs.

Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria.

The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in human mitochondria. In vitro reconstitution experiments show that GRSF1 promotes G4 melting that facilitates degradosome-mediated decay. Among degradosome and GRSF1 regulated transcripts we identified one that undergoes post-transcriptional modification. We show that GRSF1 proteins form a distinct qRRM group found only in vertebrates. The appearance of GRSF1 coincided with changes in the mitochondrial genome, which allows the emergence of G4-containing RNAs. We propose that GRSF1 appearance is an evolutionary adaptation enabling control of G4 RNA.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

[Dialogues between cell nuclei and mitochondria]. / Dialogi pomiedzy jadrem a mitochondriami.

Mitochondria are not only ATP producing organelles, but they play pivotal roles in apoptosis, neurodegeneration, cancer and aging. Mammalian mitochondrial genome is a small DNA molecule of about 16.5 kb, encoding less than 20 polypeptides and a set of ribosomal RNAs and tRNAs. In order to ensure proper cell functioning a continous communication between cell nucleus and mitochondria must be maintained. This review presents novel developments in the field of nucleo-mitochondrial communications. We discuss the import of regulatory cytosolic miRNAs into mitochondria, export of RNA from mitochondria, the existence of novel 3 polypeptides encoded by the mitochondrial genome and the transfer of mitochondrial DNA to nuclear genomes. Mechanisms of these processes and their significance for cellular homeostasis are poorly known and present an important challenge for molecular biology.

Yeast and human mitochondrial helicases.

Mitochondria are semiautonomous organelles which contain their own genome. Both maintenance and expression of mitochondrial DNA require activity of RNA and DNA helicases. In Saccharomyces cerevisiae the nuclear genome encodes four DExH/D superfamily members (MSS116, SUV3, MRH4, IRC3) that act as helicases and/or RNA chaperones. Their activity is necessary for mitochondrial RNA splicing, degradation, translation and genome maintenance. In humans the ortholog of SUV3 (hSUV3, SUPV3L1) so far is the best described mitochondrial RNA helicase. The enzyme, together with the matrix-localized pool of PNPase (PNPT1), forms an RNA-degrading complex called the mitochondrial degradosome, which localizes to distinct structures (D-foci). Global regulation of mitochondrially encoded genes can be achieved by changing mitochondrial DNA copy number. This way the proteins involved in its replication, like the Twinkle helicase (c10orf2), can indirectly regulate gene expression. Here, we describe yeast and human mitochondrial helicases that are directly involved in mitochondrial RNA metabolism, and present other helicases that participate in mitochondrial DNA replication and maintenance. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci.

RNA decay is usually mediated by protein complexes and can occur in specific foci such as P-bodies in the cytoplasm of eukaryotes. In human mitochondria nothing is known about the spatial organization of the RNA decay machinery, and the ribonuclease responsible for RNA degradation has not been identified. We demonstrate that silencing of human polynucleotide phosphorylase (PNPase) causes accumulation of RNA decay intermediates and increases the half-life of mitochondrial transcripts. A combination of fluorescence lifetime imaging microscopy with Förster resonance energy transfer and bimolecular fluorescence complementation (BiFC) experiments prove that PNPase and hSuv3 helicase (Suv3, hSuv3p and SUPV3L1) form the RNA-degrading complex in vivo in human mitochondria. This complex, referred to as the degradosome, is formed only in specific foci (named D-foci), which co-localize with mitochondrial RNA and nucleoids. Notably, interaction between PNPase and hSuv3 is essential for efficient mitochondrial RNA degradation. This provides indirect evidence that degradosome-dependent mitochondrial RNA decay takes place in foci.

RNA degradation in yeast and human mitochondria.

Expression of mitochondrially encoded genes must be finely tuned according to the cell's requirements. Since yeast and human mitochondria have limited possibilities to regulate gene expression by altering the transcription initiation rate, posttranscriptional processes, including RNA degradation, are of great importance. In both organisms mitochondrial RNA degradation seems to be mostly depending on the RNA helicase Suv3. Yeast Suv3 functions in cooperation with Dss1 ribonuclease by forming a two-subunit complex called the mitochondrial degradosome. The human ortholog of Suv3 (hSuv3, hSuv3p, SUPV3L1) is also indispensable for mitochondrial RNA decay but its ribonucleolytic partner has so far escaped identification. In this review we summarize the current knowledge about RNA degradation in human and yeast mitochondria. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Human Suv3 protein reveals unique features among SF2 helicases.

Suv3 is a helicase that is involved in efficient turnover and surveillance of RNA in eukaryotes. In vitro studies show that human Suv3 (hSuv3) in complex with human polynucleotide phosphorylase has RNA degradosome activity. The enzyme is mainly localized in mitochondria, but small fractions are found in cell nuclei. Here, two X-ray crystallographic structures of human Suv3 in complex with AMPPNP, a nonhydrolysable analog of ATP, and with a short five-nucleotide strand of RNA are presented at resolutions of 2.08 and 2.9 Å, respectively. The structure of the enzyme is very similar in the two complexes and consists of four domains. Two RecA-like domains form the tandem typical of all helicases from the SF2 superfamily which together with the C-terminal all-helical domain makes a ring structure through which the nucleotide strand threads. The mostly helical N-terminal domain is positioned externally with respect to the core of the enzyme. Most of the typical helicase motifs are present in hSuv3, but the protein shows certain unique characteristics, suggesting that Suv3 enzymes may constitute a separate subfamily of helicases.

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

The hSuv3 (human Suv3) helicase has been shown to be a major player in mitochondrial RNA surveillance and decay, but its physiological role might go beyond this functional niche. hSuv3 has been found to interact with BLM (Bloom's syndrome protein) and WRN (Werner's syndrome protein), members of the RecQ helicase family involved in multiple DNA metabolic processes, and in protection and stabilization of the genome. In the present study, we have addressed the possible role of hSuv3 in genome maintenance by examining its potential association with key interaction partners of the RecQ helicases. By analysis of hSuv3 co-IP (co-immunoprecipitation) complexes, we identify two new interaction partners of hSuv3: the RPA (replication protein A) and FEN1 (flap endonuclease 1). Utilizing an in vitro biochemical assay we find that low amounts of RPA inhibit helicase activity of hSuv3 on a forked substrate. Another single-strand-binding protein, mtSSB (mitochondrial single-strand-binding protein), fails to affect hSuv3 activity, indicating that the functional interaction is specific for hSuv3 and RPA. Further in vitro studies demonstrate that the flap endonuclease activity of FEN1 is stimulated by hSuv3 independently of flap length. hSuv3 is generally thought to be a mitochondrial helicase, but the physical and functional interactions between hSuv3 and known RecQ helicase-associated proteins strengthen the hypothesis that hSuv3 may play a significant role in nuclear DNA metabolism as well.

Involvement of human ELAC2 gene product in 3' end processing of mitochondrial tRNAs.

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.

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L.

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine-subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R-like enzyme, which possesses both processive exo- and endonuclease activities, whereas the latter is a distributive RNase D-like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs--hDIS3 and hDIS3L--with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.

Assays of the helicase, ATPase, and exoribonuclease activities of the yeast mitochondrial degradosome.

The mitochondrial degradosome (mtEXO) is the main enzymatic complex in RNA degradation, processing, and surveillance in Saccharomyces cerevisiae mitochondria. It consists of two nuclear-encoded subunits: the ATP-dependent RNA helicase Suv3p and the 3' to 5' exoribonuclease Dss1p. The two subunits depend on each other for their activity; the complex can therefore be considered as a model system for the cooperation of RNA helicases and exoribonucleases in RNA degradation. All the three activities of the complex (helicase, ATPase, and exoribonuclease) can be studied in vitro using recombinant proteins and protocols presented in this chapter.

RNA turnover in human mitochondria: more questions than answers?

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.

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

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.

In vivo and in vitro approaches for studying the yeast mitochondrial RNA degradosome complex.

The mitochondrial degradosome (mtEXO) of S. cerevisiae is the main exoribonuclease of yeast mitochondria. It is involved in many pathways of mitochondrial RNA metabolism, including RNA degradation, surveillance, and processing, and its activity is essential for mitochondrial gene function. The mitochondrial degradosome is a very simple example of a 3' to 5'-exoribonucleolytic complex. It is composed of only two subunits: Dss1p, which is an RNR (RNase II-like) family exoribonuclease, and Suv3p, which is a DExH/D-box RNA helicase. The two subunits form a tight complex and their activities are highly interdependent, with the RNase activity greatly enhanced in the presence of the helicase subunit, and the helicase activity entirely dependent on the presence of the ribonuclease subunit. In this chapter, we present methods for studying the function of the yeast mitochondrial degradosome in vivo, through the analysis of degradosome-deficient mutant yeast strains, and in vitro, through heterologous expression in E. coli and purification of the degradosome subunits and reconstitution of a functional complex. We provide the protocols for studying ribonuclease, ATPase, and helicase activities and for measuring the RNA binding capacity of the complex and its subunits.

Interaction of human SUV3 RNA/DNA helicase with BLM helicase; loss of the SUV3 gene results in mouse embryonic lethality.

The SUV3 gene is present in all eukaryotes and encodes an RNA/DNA helicase which operates both in mitochondria and cell nuclei. To assess its function in mammals we generated a mouse mutant strain in which the 3' part of the SUV3 gene is disrupted. The mutated allele is a hypomorph transmitted from one generation to another at a frequency about 35% lower than expected while mice homozygous for the mutation die in utero before midgestation. Using ELISA binding assays we show that human SUV3 protein interacts with human WRN and BLM helicases. The binding to BLM protein was 10-fold stronger (with a K(d) of 0.5nM) than to WRN protein (K(d) of 5nM). Silencing of the SUV3 gene in the human cell line HeLa resulted in elevation of homologous recombination as measured by the frequency of sister chromatid exchange during mitotic cell division. These results indicate that the SUV3 protein is required in mammalian development and in somatic cells participates in genome maintenance through interaction with other genome fidelity housekeepers.

In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence.

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.

Down-regulation of human RNA/DNA helicase SUV3 induces apoptosis by a caspase- and AIF-dependent pathway.

BACKGROUND INFORMATION: The nuclear gene hSUV3 (human SUV3) encodes an ATP-dependent DNA/RNA helicase. In the yeast Saccharomyces cerevisiae the orthologous Suv3 protein is localized in mitochondria, and is a subunit of the degradosome complex which regulates RNA surveillance and turnover. In contrast, the functions of human SUV3 are not known to date. RESULTS: In the present study, we show that a fraction of human SUV3 helicase is localized in the nucleus. Using small interfering RNA gene silencing in HeLa cells, we demonstrate that down-regulation of hSUV3 results in cell cycle perturbations and in apoptosis, which is both AIF- and caspase-dependent, and proceeds with the induction of p53. CONCLUSIONS: In addition to its mitochondrial localization, human SUV3 plays an important role in the nucleus and is probably involved in chromatin maintenance.

Prediction of the structure of the common perimitochondrial localization signal of nuclear transcripts in yeast.

Many nuclear genes encoding mitochondrial proteins require specific localization of their mRNAs to the vicinity of mitochondria for proper expression. Studies in Saccharomyces cerevisiae have shown that the cis-acting signal responsible for subcellular localization of mRNAs is localized in the 3' UTR of the transcript. In this paper we present an in silico approach for prediction of a common perimitochondrial localization signal of nuclear transcripts encoding mitochondrial proteins. We computed a consensus structure for this signal by comparison of 3' UTR models for about 3000 yeast transcripts with known localization. Our studies show a short stem-loop structure which appears in most mRNAs localized to the vicinity of mitochondria. The degree of similarity of a given 3' UTR to our consensus structure strongly correlates with experimentally determined perimitochondrial localization of the mRNA, therefore we believe that the structure we predicted acts as a subcellular localization signal. Since our algorithm operates on structures, it seems to be more reliable than sequence-based algorithms. The good predictive value of our model is supported by statistical analysis.