Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

The tumor suppressor microRNA let-7 inhibits human LINE-1 retrotransposition.

Nearly half of the human genome is made of transposable elements (TEs) whose activity continues to impact its structure and function. Among them, Long INterspersed Element class 1 (LINE-1 or L1) elements are the only autonomously active TEs in humans. L1s are expressed and mobilized in different cancers, generating mutagenic insertions that could affect tumor malignancy. Tumor suppressor microRNAs are ∼22nt RNAs that post-transcriptionally regulate oncogene expression and are frequently downregulated in cancer. Here we explore whether they also influence L1 mobilization. We show that downregulation of let-7 correlates with accumulation of L1 insertions in human lung cancer. Furthermore, we demonstrate that let-7 binds to the L1 mRNA and impairs the translation of the second L1-encoded protein, ORF2p, reducing its mobilization. Overall, our data reveals that let-7, one of the most relevant microRNAs, maintains somatic genome integrity by restricting L1 retrotransposition.

sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay.

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

LINE-1 Evasion of Epigenetic Repression in Humans.

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.

The impact of transposable element activity on therapeutically relevant human stem cells.

Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving loss of motor neurons and having no known cure and uncertain etiology. Several studies have drawn connections between altered retrotransposon expression and ALS. Certain features of the LINE-1 (L1) retrotransposon-encoded ORF1 protein (ORF1p) are analogous to those of neurodegeneration-associated RNA-binding proteins, including formation of cytoplasmic aggregates. In this study we explore these features and consider possible links between L1 expression and ALS. RESULTS: We first considered factors that modulate aggregation and subcellular distribution of LINE-1 ORF1p, including nuclear localization. Changes to some ORF1p amino acid residues alter both retrotransposition efficiency and protein aggregation dynamics, and we found that one such polymorphism is present in endogenous L1s abundant in the human genome. We failed, however, to identify CRM1-mediated nuclear export signals in ORF1p nor strict involvement of cell cycle in endogenous ORF1p nuclear localization in human 2102Ep germline teratocarcinoma cells. Some proteins linked with ALS bind and colocalize with L1 ORF1p ribonucleoprotein particles in cytoplasmic RNA granules. Increased expression of several ALS-associated proteins, including TAR DNA Binding Protein (TDP-43), strongly limits cell culture retrotransposition, while some disease-related mutations modify these effects. Using quantitative reverse transcription PCR (RT-qPCR) of ALS tissues and reanalysis of publicly available RNA-Seq datasets, we asked if changes in expression of retrotransposons are associated with ALS. We found minimal altered expression in sporadic ALS tissues but confirmed a previous report of differential expression of many repeat subfamilies in C9orf72 gene-mutated ALS patients. CONCLUSIONS: Here we extended understanding of the subcellular localization dynamics of the aggregation-prone LINE-1 ORF1p RNA-binding protein. However, we failed to find compelling evidence for misregulation of LINE-1 retrotransposons in sporadic ALS nor a clear effect of ALS-associated TDP-43 protein on L1 expression. In sum, our study reveals that the interplay of active retrotransposons and the molecular features of ALS are more complex than anticipated. Thus, the potential consequences of altered retrotransposon activity for ALS and other neurodegenerative disorders are worthy of continued investigation.

Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. About 90% of ALS cases are without a known genetic cause. The human endogenous retrovirus multi-copy HERV-K(HML-2) group was recently reported to potentially contribute to neurodegeneration and disease pathogenesis in ALS because of transcriptional upregulation and toxic effects of HML-2 Envelope (Env) protein. Env and other proteins are encoded by some transcriptionally active HML-2 loci. However, more detailed information is required regarding which HML-2 loci are transcribed in ALS, which of their proteins are expressed, and differences between the disease and non-disease states. METHODS: For brain and spinal cord tissue samples from ALS patients and controls, we identified transcribed HML-2 loci by generating and mapping HML-2-specific cDNA sequences. We predicted expression of HML-2 env gene-derived proteins based on the observed cDNA sequences. Furthermore, we determined overall HML-2 transcript levels by RT-qPCR and investigated presence of HML-2 Env protein in ALS and control tissue samples by Western blotting. RESULTS: We identified 24 different transcribed HML-2 loci. Some of those loci are transcribed at relatively high levels. However, significant differences in HML-2 loci transcriptional activities were not seen when comparing ALS and controls. Likewise, overall HML-2 transcript levels, as determined by RT-qPCR, were not significantly different between ALS and controls. Indeed, we were unable to detect full-length HML-2 Env protein in ALS and control tissue samples despite reasonable sensitivity. Rather our analyses suggest that a number of HML-2 protein variants other than full-length Env may potentially be expressed in ALS patients. CONCLUSIONS: Our results expand and refine recent publications on HERV-K(HML-2) and ALS. Some of our results are in conflict with recent findings and call for further specific analyses. Our profiling of HML-2 transcription in ALS opens up the possibility that HML-2 proteins other than canonical full-length Env may have to be considered when studying the role of HML-2 in ALS disease.

RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition.

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.

Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor.

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.

Control of mammalian retrotransposons by cellular RNA processing activities.

Retrotransposons make up roughly 50% of the mammalian genome and have played an important role in genome evolution. A small fraction of non-LTR retrotransposons, LINE-1 and SINE elements, is currently active in the human genome. These elements move in our genome using an intermediate RNA and a reverse transcriptase activity by a copy and paste mechanism. Their ongoing mobilization can impact the human genome leading to several human disorders. However, how the cell controls the activity of these elements minimizing their mutagenic effect is not fully understood. Recent studies have highlighted that the intermediate RNA of retrotransposons is a target of different mechanisms that limit the mobilization of endogenous retrotransposons in mammals. Here, we provide an overview of recent discoveries that show how RNA processing events can act to control the activity of mammalian retrotransposons and discuss several arising questions that remain to be answered.

The translational landscape of the splicing factor SRSF1 and its role in mitosis.

The shuttling Serine/Arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1,500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets; strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.

The Microprocessor controls the activity of mammalian retrotransposons.

More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.

Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon.

It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3' terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts.

L1Tc is the best represented autonomous LINE of the Trypanosoma cruzi genome, throughout which several functional copies may exist. In this study, we show that the first 77 bp of L1Tc (Pr77) (also present in the T. cruzi non-autonomous retrotransposon NARTc, in the Trypanosoma brucei RIME/ingi elements, and in the T. cruzi, T. brucei and Leishmania major degenerate L1Tc/ingi-related elements [DIREs]) behave as a promoter element that activates gene transcription. The transcription rate promoted by Pr77 is 10-14-fold higher than that mediated by sequences located upstream from the T. cruzi tandemly repeated genes KMP11 and the GAPDH. The Pr77 promoter-derived mRNAs initiate at nucleotide +1 of L1Tc, are unspliced and translated. L1Tc transcripts show a moderate half life and are RNA pol II dependent. The presence of an internal promoter at the 5' end of L1Tc favors the production of full-length L1Tc RNAs and reinforces the hypothesis that this mobile element may be naturally autonomous in its transposition.

The L1Tc C-terminal domain from Trypanosoma cruzi non-long terminal repeat retrotransposon codes for a protein that bears two C2H2 zinc finger motifs and is endowed with nucleic acid chaperone activity.

L1Tc, a non-long terminal repeat retrotransposon from Trypanosoma cruzi, is a 4.9-kb actively transcribed element which contains a single open reading frame coding for the machinery necessary for its autonomous retrotransposition. In this paper, we analyze the protein encoded by the L1Tc 3' region, termed C2-L1Tc, which contains two zinc finger motifs similar to those present in the TFIIIA transcription factor family. C2-L1Tc binds nucleic acids with different affinities, such that RNA > tRNA > single-stranded DNA > double-stranded DNA, without any evidence for sequence specificity. C2-L1Tc also exhibits nucleic acid chaperone activity on different DNA templates that may participate in the mechanism of retrotransposition of the element. C2-L1Tc promotes annealing of complementary oligonucleotides, prevents melting of perfect DNA duplexes, and facilitates the strand exchange between DNAs to form the most stable duplex DNA in competitive displacement assays. Mapping of regions of C2-L1Tc using specific peptides showed that nucleic acid chaperone activity required a short basic sequence accompanied by a zinc finger motif or by another basic region such as RRR. Thus, a short basic polypeptide containing the two C(2)H(2) motifs promotes formation of the most stable duplex DNA at a concentration only three times higher than that required for C2-L1Tc.

Identification of non-autonomous non-LTR retrotransposons in the genome of Trypanosoma cruzi.

As observed for most eukaryotic cells, trypanosomatids contains non-LTR retrotransposons randomly inserted in the nuclear genome. Autonomous retroelements which, code for their own transposition, have been characterized in Trypanosoma brucei (ingi) and Trypanosoma cruzi (L1Tc), whereas non-autonomous retroelements have only been characterized in T. brucei (RIME). Here, we have characterized in the genome of Trypanosoma cruzi four complete copies of a non-autonomous non-LTR retrotransposon, called NARTc. This 0.26 kb NARTc element has the characteristics of non-LTR retrotransposons: the presence a poly(dA) tail and of a short flanking duplicated motif. Analysis of the Genome Survey Sequence databases indicated that the Trypanosoma cruzi haploid genome contains about 140 NARTc copies and about twice as many L1Tc copies. Interestingly, the NARTc and L1Tc retroelements share, with the Trypanosoma brucei ingi and RIME retrotransposons, a common sequence (the first 45 bp with 91% identity), whereas the remaining sequences are very divergent. This suggests that these four trypanosome non-LTR retrotransposons were derived from the same common ancester and the sequence of their 5'-extremity may have a functional role. In addition, the genome of Leishmania major contains the same conserved motif present in the trypanosome retroelements, whicle no transposable elements have been detected so far in Leishmania sp.

The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.

The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of the Trypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His(6)-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [(3)H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [(32)P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg(2+)-dependent, and it is also active in the presence of the Mn(2+) ion. The optimal condition of RNase H activity is found at pH 8 and 37 degrees C, although it also has significant enzymatic activity at 19 degrees C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.