Dr. Francisco Jose Sanchez-Luque, currently a Research Fellow in the Genome Plasticity and Disease (GDP) group, in Mater Research Institute (MRI), Brisbane area (Australia), from February 2015.
He is currently funded by the European Council through a Marie Curie-International Outgoing Fellowship (Seventh Framework Programme, Marie Curie Actions). Technically based in the group of ‘Biology of LINE-1 Retrotransposons’, in Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncologic Research (Genyo), Granada (Spain), his project comprises an outgoing phase in the GPD group at Mater as a secondment. This outgoing phase is currently ongoing.read more
Dr. Sanchez-Luque obtained the bachelor degree in Biology in the University of Cordoba (Spain), in 2003, where he was awarded the Extraordinary Prize of Degree in 2004. During this time, he specialized in Molecular Biology and underwent research activities in the departments of Microbiology and Cellular Biology, Physiology and Immunology. Partially overlapping with the Bachelor Degree, he performed two research stays in the laboratory of Dr. Joaquin Teixido working on chemokines and cell migration in the Centre of Biological Research, from the Spanish National Research Council (CSIC), Madrid (Spain).
Dr. Sanchez-Luque PhD is titled ‘Inhibitor RNAs against Human Immunodeficiency Virus’ (HIV) and was developed in the laboratory of Dr. Alfredo Berzal-Herranz, in the Institute of Parasitology and Biomedicine ‘Lopez-Neyra’ (IPB-LN), from CSIC, Granada (Spain). This involved the use of ribozymes, aptamers and natural antisense RNAs as functional substrates to engineer RNAs targeting the genomic RNA of HIV either to destroy or to disable some of its functional domains. Here he developed a strong interest in the research in functional RNA molecules and their link with the origin of the life. During the PhD period, Dr. Sanchez-Luque performed a short stay in the Laboratory of Retrovirology, under supervision of Prof. Ben Berkhout, in the Academic Medical Centre (AMC) of the University of Amsterdam in The Netherlands. The major achievements of this work were the contribution to the elucidation of the mechanistic model for HIV RNA dimerization and the development of a short (16nt) inhibitor RNA that was subject of a patent. The latter was product of a collaboration established with Dr. Carlos Briones, from the Centre of Astrobiology (CAB) (CSIC-INTA), which combined experimentally obtained RNA molecules population and bioinformatic identification and refinement of a functional RNA motif inhibiting HIV. Dr. Sanchez-Luque’s PhD was funded by a ‘university professorate training fellowship’ from the extinct Spanish Ministry of Education and Culture (MEC) from 2005 to 2008.
Dr. Sanchez-Luque moved to the laboratory of Dr. Manuel C. Lopez and Dr. M. Carmen Thomas from 2010 to 2014, also at the IPB-LN. There, he first contacted the research in mobile elements, studying the retrotransposon L1Tc in Trypanosoma cruzi, causative agent of Chagas disease. Here, he combined his experience in functional RNAs and described the presence of a rare ribozyme in the 5′ end of the RNA of this retrotransposon. This ribozyme was further characterized in other related elements in Trypanosomatids, like NARTc (in T. cruzi), L1Tco and NARTco (in T. congolense) as well as different SIDER elements in T. vivax and Leishmania spp. During this period, Dr. Sanchez-Luque’s research was funded by the Cooperative Research Network in Tropical Diseases (RICET).
On February 2014, Dr. Sanchez-Luque started as a Research Officer in the GPD group at MRI, co-funded by the Spanish Foundation Martin Escudero and MRI. The major aim during this period was the research on the activity of mobile genetic elements (particularly LINE-1 or L1) during the neurogenesis in the adult brain, and its possible relation with some neurological diseases like Rett syndrome. In 2015, Dr. Sanchez-Luque was upgraded to Research Fellow since he obtained a Marie Curie-International Outgoing Fellowship, and started as a secondment from Genyo. During 2015, Dr. Sanchez-Luque has contributed to the elucidation of the transposition rate of the L1 in the neurogenic regions of the human brain and is currently working to understand the impact of this somatic genome reshaping carried out by L1 in the brain function.
Ewing, Adam D; Smits, Nathan; Sanchez-Luque, Francisco J; Faivre, Jamila; Brennan, Paul M; Richardson, Sandra R; Cheetham, Seth W; Faulkner, Geoffrey J
In: Molecular Cell, 2020, ISSN: 1097-2765.
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Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.
Salvador-Palomeque, Carmen; Sanchez-Luque, Francisco J; Fortuna, Patrick R J; Ewing, Adam D; Wolvetang, Ernst J; Richardson, Sandra R; Faulkner, Geoffrey J
In: Mol Cell Biol, 39 (7), 2019, ISSN: 0270-7306.
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The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells., The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. L1 retrotransposition can also occur in the neuronal lineage and cause somatic mosaicism. Although DNA methylation mediates L1 promoter repression, the temporal pattern of methylation applied to individual RC-L1s during neurogenesis is unclear. Here, we identified a de novo L1 insertion in a human induced pluripotent stem cell (hiPSC) line via retrotransposon capture sequencing (RC-seq). The L1 insertion was full-length and carried 5ʹ and 3ʹ transductions. The corresponding donor RC-L1 was part of a large and recently active L1 transduction family and was highly mobile in a cultured-cell L1 retrotransposition reporter assay. Notably, we observed distinct and dynamic DNA methylation profiles for the de novo L1 and members of its extended transduction family during neuronal differentiation. These experiments reveal how a de novo L1 insertion in a pluripotent stem cell is rapidly recognized and repressed, albeit incompletely, by the host genome during neurodifferentiation, while retaining potential for further retrotransposition.
Sanchez-Luque, Francisco J; Kempen, Marie-Jeanne H C; Gerdes, Patricia; Vargas-Landin, Dulce B; Richardson, Sandra R; Troskie, Robin-Lee; Jesuadian, Samuel J; Cheetham, Seth W; Carreira, Patricia E; Salvador-Palomeque, Carmen; García-Cañadas, Marta; Muñoz-Lopez, Martin; Sanchez, Laura; Lundberg, Mischa; Macia, Angela; Heras, Sara R; Brennan, Paul M; Lister, Ryan; Garcia-Perez, Jose L; Ewing, Adam D; Faulkner, Geoffrey J
LINE-1 Evasion of Epigenetic Repression in Humans (Journal Article)
In: Molecular Cell, 0 (0), 2019, ISSN: 1097-2765.
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textlessh2textgreaterSummarytextless/h2textgreatertextlessptextgreaterEpigenetic 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 3ʹ transduction. The source (donor) L1 for this insertion was slightly 5ʹ truncated, lacked the YY1 binding site, and was highly mobile when tested textitin 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.textless/ptextgreater
Nguyen, Thu H M; Carreira, Patricia E; Sanchez-Luque, Francisco J; Schauer, Stephanie N; Fagg, Allister C; Richardson, Sandra R; Davies, Claire M; Jesuadian, Samuel J; Kempen, Marie-Jeanne H C; Troskie, Robin-Lee; James, Cini; Beaven, Elizabeth A; Wallis, Tristan P; Coward, Jermaine I G; Chetty, Naven P; Crandon, Alexander J; Venter, Deon J; Armes, Jane E; Perrin, Lewis C; Hooper, John D; Ewing, Adam D; Upton, Kyle R; Faulkner, Geoffrey J
L1 Retrotransposon Heterogeneity in Ovarian Tumor Cell Evolution (Journal Article)
In: Cell Reports, 23 (13), pp. 3730–3740, 2018, ISSN: 2211-1247.
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LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity.
Schauer, Stephanie N; Carreira, Patricia E; Shukla, Ruchi; Gerhardt, Daniel J; Gerdes, Patricia; Sanchez-Luque, Francisco J; Nicoli, Paola; Kindlova, Michaela; Ghisletti, Serena; Santos, Alexandre Dos; Rapoud, Delphine; Samuel, Didier; Faivre, Jamila; Ewing, Adam D; Richardson, Sandra R; Faulkner, Geoffrey J
In: Genome Research, 2018, ISSN: 1088-9051, 1549-5469.
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The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies. We previously employed retrotransposon capture sequencing (RC-seq) to analyze hepatocellular carcinoma (HCC) samples from patients infected with hepatitis B or hepatitis C virus and identified L1 variants responsible for activating oncogenic pathways. Here, we have applied RC-seq and whole-genome sequencing (WGS) to an Abcb4 (Mdr2)−/− mouse model of hepatic carcinogenesis and demonstrated for the first time that L1 mobilization occurs in murine tumors. In 12 HCC nodules obtained from 10 animals, we validated four somatic L1 insertions by PCR and capillary sequencing, including TF subfamily elements, and one GF subfamily example. One of the TF insertions carried a 3′ transduction, allowing us to identify its donor L1 and to demonstrate that this full-length TF element retained retrotransposition capacity in cultured cancer cells. Using RC-seq, we also identified eight tumor-specific L1 insertions from 25 HCC patients with a history of alcohol abuse. Finally, we used RC-seq and WGS to identify three tumor-specific L1 insertions among 10 intra-hepatic cholangiocarcinoma (ICC) patients, including one insertion traced to a donor L1 on Chromosome 22 known to be highly active in other cancers. This study reveals L1 mobilization as a common feature of hepatocarcinogenesis in mammals, demonstrating that the phenomenon is not restricted to human viral HCC etiologies and is encountered in murine liver tumors.
Richardson, Sandra R; Gerdes, Patricia; Gerhardt, Daniel J; Sanchez-Luque, Francisco J; Bodea, Gabriela-Oana; noz-Lopez, Martin Mu; Jesuadian, Samuel J; Kempen, Marie-Jeanne H C; Carreira, Patricia E; Jeddeloh, Jeffrey A; Garcia-Perez, Jose L; Jr, Haig H Kazazian; Ewing, Adam D; Faulkner, Geoffrey J
In: Genome Res., 27 (8), pp. 1395–1405, 2017.
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LINE-1 (L1) retrotransposons are a noted source of genetic
diversity and disease in mammals. To expand its genomic
footprint, L1 must mobilize in cells that will contribute
their genetic material to subsequent generations. Heritable L1
insertions may therefore arise in germ cells and in
pluripotent embryonic cells, prior to germline specification,
yet the frequency and predominant developmental timing of such
events remain unclear. Here, we applied mouse retrotransposon
capture sequencing (mRC-seq) and whole-genome sequencing (WGS)
to pedigrees of C57BL/6J animals, and uncovered an L1
insertion rate of $geq$1 event per eight births. We traced
heritable L1 insertions to pluripotent embryonic cells and,
strikingly, to early primordial germ cells (PGCs). New L1
insertions bore structural hallmarks of target-site primed
reverse transcription (TPRT) and mobilized efficiently in a
cultured cell retrotransposition assay. Together, our results
highlight the rate and evolutionary impact of heritable L1
retrotransposition and reveal retrotransposition-mediated
genomic diversification as a fundamental property of
pluripotent embryonic cells in vivo.
Sanchez-Luque, Francisco J; Richardson, Sandra R; Faulkner, Geoffrey J
Analysis of Somatic LINE-1 Insertions in Neurons (Incollection)
In: Genomic Mosaicism in Neurons and Other Cell Types, pp. 219–251, Humana Press, New York, NY, 2017, ISBN: 978-1-4939-7279-1 978-1-4939-7280-7, (DOI: 10.1007/978-1-4939-7280-7_12).
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The method described here is designed to detect and localize somatic genome variation caused by the human retrotransposon LINE-1 (L1) in the genome of neuronal cells. This method combines single-cell manipulation and whole genome amplification technology with a hybridization-based, high-throughput sequencing method called Retrotransposon Capture sequencing (RC-seq) for the precise analysis of the L1 insertion content of single cell genomes. The method is divided into four major sections: extraction of neuronal nuclei and single nuclei isolation; whole genome amplification; RC-seq; and experimental validation of putative insertions.
Sanchez-Luque, Francisco J; Richardson, Sandra R; Faulkner, Geoffrey J
In: Methods Mol. Biol., 1400 , pp. 47–77, 2016.
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Mobile genetic elements (MGEs) are of critical importance in
genomics and developmental biology. Polymorphic and somatic
MGE insertions have the potential to impact the phenotype of
an individual, depending on their genomic locations and
functional consequences. However, the identification of
polymorphic and somatic insertions among the plethora of
copies residing in the genome presents a formidable technical
challenge. Whole genome sequencing has the potential to
address this problem; however, its efficacy depends on the
abundance of cells carrying the new insertion. Robust
detection of somatic insertions present in only a subset of
cells within a given sample can also be prohibitively
expensive due to a requirement for high sequencing depth.
Here, we describe retrotransposon capture sequencing (RC-seq),
a sequence capture approach in which Illumina libraries are
enriched for fragments containing the 5' and 3' termini of
specific MGEs. RC-seq allows the detection of known
polymorphic insertions present in an individual, as well as
the identification of rare or private germline insertions not
previously described. Furthermore, RC-seq can be used to
detect and characterize somatic insertions, providing a
valuable tool to elucidate the extent and characteristics of
MGE activity in healthy tissues and in various disease states.
Sanchez-Luque, Francisco J; Richardson, Sandra R; Faulkner, Geoffrey J
In: Methods in Molecular Biology (Clifton, N.J.), 1400 , pp. 47–77, 2016, ISSN: 1940-6029.
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Mobile genetic elements (MGEs) are of critical importance in genomics and developmental biology. Polymorphic and somatic MGE insertions have the potential to impact the phenotype of an individual, depending on their genomic locations and functional consequences. However, the identification of polymorphic and somatic insertions among the plethora of copies residing in the genome presents a formidable technical challenge. Whole genome sequencing has the potential to address this problem; however, its efficacy depends on the abundance of cells carrying the new insertion. Robust detection of somatic insertions present in only a subset of cells within a given sample can also be prohibitively expensive due to a requirement for high sequencing depth. Here, we describe retrotransposon capture sequencing (RC-seq), a sequence capture approach in which Illumina libraries are enriched for fragments containing the 5' and 3' termini of specific MGEs. RC-seq allows the detection of known polymorphic insertions present in an individual, as well as the identification of rare or private germline insertions not previously described. Furthermore, RC-seq can be used to detect and characterize somatic insertions, providing a valuable tool to elucidate the extent and characteristics of MGE activity in healthy tissues and in various disease states.
Upton, Kyle R; Gerhardt, Daniel J; Jesuadian, Samuel J; Richardson, Sandra R; Sánchez-Luque, Francisco J; Bodea, Gabriela O; Ewing, Adam D; Salvador-Palomeque, Carmen; van der Knaap, Marjo S; Brennan, Paul M; Vanderver, Adeline; Faulkner, Geoffrey J
Ubiquitous L1 mosaicism in hippocampal neurons (Journal Article)
In: Cell, 161 (2), pp. 228–239, 2015.
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Somatic LINE-1 (L1) retrotransposition during neurogenesis is
a potential source of genotypic variation among neurons. As a
neurogenic niche, the hippocampus supports pronounced L1
activity. However, the basal parameters and biological impact
of L1-driven mosaicism remain unclear. Here, we performed
single-cell retrotransposon capture sequencing (RC-seq) on
individual human hippocampal neurons and glia, as well as
cortical neurons. An estimated 13.7 somatic L1 insertions
occurred per hippocampal neuron and carried the sequence
hallmarks of target-primed reverse transcription. Notably,
hippocampal neuron L1 insertions were specifically enriched in
transcribed neuronal stem cell enhancers and hippocampus
genes, increasing their probability of functional relevance.
In addition, bias against intronic L1 insertions sense
oriented relative to their host gene was observed, perhaps
indicating moderate selection against this configuration in
vivo. These experiments demonstrate pervasive L1 mosaicism at
genomic loci expressed in hippocampal neurons.
Reyes-Darias, José A; Sánchez-Luque, Francisco J; Morales, Juan Carlos; Pérez-Rentero, Sonia; Eritja, Ramón; Berzal-Herranz, Alfredo
In: Chembiochem: A European Journal of Chemical Biology, 16 (4), pp. 584–591, 2015, ISSN: 1439-7633.
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Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.
Sánchez-Luque, Francisco J; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo
In: Scientific Reports, 4 , pp. 6242, 2014, ISSN: 2045-2322.
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The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.
Sánchez-Luque, Francisco José; López, Manuel Carlos; Carreira, Patricia Eugenia; Alonso, Carlos; Thomas, María Carmen
In: BMC genomics, 15 , pp. 340, 2014, ISSN: 1471-2164.
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BACKGROUND: Hepatitis Delta Virus (HDV)-like ribozymes have recently been found in many mobile elements in which they take part in a mechanism that releases intermediate RNAs from cellular co-transcripts. L1Tc in Trypanosoma cruzi is one of the elements in which such a ribozyme is located. It lies in the so-called Pr77-hallmark, a conserved region shared by retrotransposons belonging to the trypanosomatid L1Tc/ingi clade. The wide distribution of the Pr77-hallmark detected in trypanosomatid retrotransposons renders the potential catalytic activity of these elements worthy of study: their distribution might contribute to host genetic regulation at the mRNA level. Indeed, in Leishmania spp, the pervasive presence of these HDV-like ribozyme-containing mobile elements in certain 3'-untranslated regions of protein-coding genes has been linked to mRNA downregulation.
RESULTS: Intensive screening of publicly available trypanosomatid genomes, combined with manual folding analyses, allowed the isolation of putatively Pr77-hallmarks with HDV-like ribozyme activity. This work describes the conservation of an HDV-like ribozyme structure in the Pr77 sequence of retrotransposons in a wide range of trypanosomatids, the catalytic function of which is maintained in the majority.These results are consistent with the previously suggested common phylogenetic origin of the elements that belong to this clade, although in some cases loss of functionality appears to have occurred and/or perhaps molecular domestication by the host.
CONCLUSIONS: These HDV-like ribozymes are widely distributed within retrotransposons across trypanosomatid genomes. This type of ribozyme was once thought to be rare in nature, but in fact it would seem to be abundant in trypanosomatid transcripts. It can even form part of the pool of mRNA 3'-untranslated regions, particularly in Leishmania spp. Its putative regulatory role in host genetic expression is discussed.
Reyes-Darias, José A; Sánchez-Luque, Francisco J; Berzal-Herranz, Alfredo
In: Virus Research, 169 (1), pp. 63–71, 2012, ISSN: 1872-7492.
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The HIV-1 genome consists of two identical RNA molecules non-covalently linked by their 5' unstranslatable regions (5' UTR). The high level of sequence and structural conservation of this region correlates with its important functional involvement in the viral cycle, making it an attractive target for antiviral treatments based on antisense technology. Ten unmodified DNA antisense oligonucleotides (ODNs) targeted against different conserved structural elements within the 5' UTR were assayed for their capacity to interfere with HIV-1 RNA dimerisation, inhibit gene expression, and prevent virus production in cell cultures. The results show that, in addition to the well-characterised dimerisation initiation site (DIS), targeting of the AUG-containing structural element may reflect its direct role in HIV-1 genomic RNA dimerisation in vitro. Similarly, blocking the 3' end sequences of the stem-loop domain containing the primer biding site interferes with RNA dimerisation. Targeting the apical portion of the TAR element, however, appears to promote dimerisation. ODNs targeted against the conserved polyadenylation signal [Poly(A)], the primer binding site (PBS), the major splicing donor (SD) or the major packaging signal (Psi), and AUG-containing structural elements led to a highly efficient inhibition of HIV-1 gene expression and virus production in cell culture. Together, these results support the idea that ODNs possess great potential as molecular tools for the functional characterisation of viral RNA structural domains. Moreover, the targeting of these domains leads to the potent inhibition of viral replication, underscoring the potential of conserved structural RNA elements as antiviral targets.
Sánchez-Luque, Francisco J; López, Manuel C; Macias, Francisco; Alonso, Carlos; Thomas, Carmen M
In: Nucleic Acids Research, 39 (18), pp. 8065–8077, 2011, ISSN: 1362-4962.
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L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5'-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5'-end of the 3'-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5'-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.
Sánchez-Luque, Francisco J; Reyes-Darias, José A; Puerta-Fernández, Elena; Berzal-Herranz, Alfredo
In: Molecules (Basel, Switzerland), 15 (7), pp. 4757–4772, 2010, ISSN: 1420-3049.
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The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.
Marton, Soledad; Reyes-Darias, José A; Sánchez-Luque, Francisco J; Romero-López, Cristina; Berzal-Herranz, Alfredo
In: Molecules (Basel, Switzerland), 15 (7), pp. 4610–4638, 2010, ISSN: 1420-3049.
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It was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. Quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. In vitro selection and evolution strategies have been extremely useful in the analysis of functional RNA and DNA molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize DNA and RNA molecules with potential therapeutic and diagnostic applications. The great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. This review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids.
Bartolomé, Rubén A; Wright, Natalia; Molina-Ortiz, Isabel; Sánchez-Luque, Francisco J; Teixidó, Joaquin
In: Cancer Research, 68 (20), pp. 8221–8230, 2008, ISSN: 1538-7445.
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The GTPase RhoA is a downstream target of heterotrimeric G(13) proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Galpha(13) form (G(alpha)(13)QL) or lysophosphatidylcholine (LPC)-promoted signaling through G(alpha)(13)-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing G(alpha)(13)QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream G(alpha)(13) target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of G(alpha)(13)QL melanoma cells. Importantly, G(alpha)(13)QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that G(alpha)(13)-dependent downstream effects on RhoA activation and invasion tightly depend on cell type-specific GAP activities and that G(alpha)(13)-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis.
Reyes-Darias, José A; Sánchez-Luque, Francisco J; Berzal-Herranz, Alfredo
Inhibition of HIV-1 replication by RNA-based strategies (Journal Article)
In: Current HIV research, 6 (6), pp. 500–514, 2008, ISSN: 1873-4251.
The major etiologic agent of the acquired immunodeficiency syndrome (AIDS) is the human immunodeficiency virus type 1 (HIV-1), which belongs to the family of human retroviruses. This pandemic infection affects millions of people worldwide. The most efficient current treatment regimen for HIV-infected individuals combines two or more drugs targeting different HIV-specific enzymes. However, the emergence of multiple drug-resistant HIV-1 strains and the side effects of drug-based therapies make alternative approaches for the treatment of HIV infection and AIDS necessary. RNA-based antiviral approaches are among the most promising for developing long-term anti-HIV therapies. Anti-HIV-1 RNA-based strategies include ribozymes, antisense RNAs, RNA aptamers, RNA decoys, external guide sequences (EGS) for site-specific cleavage of RNA molecules with human ribonuclease P (RNase P), modified small nuclear RNA (RNAu) and small interfering RNAs (siRNAs). This review describes the main features and functions of viral and cellular targets as well as the different classes of RNA molecules that have been explored in developing therapeutic strategies against HIV infection. Many RNA-based strategies are already being tested in human clinical trials or are currently being developed for future trials.
Barroso-DelJesus, Alicia; Puerta-Fernández, Elena; Tapia, Natalia; Romero-López, Cristina; Sánchez-Luque, Francisco José; Martínez, Miguel Angel; Berzal-Herranz, Alfredo
Inhibition of HIV-1 replication by an improved hairpin ribozyme that includes an RNA decoy (Journal Article)
In: RNA biology, 2 (2), pp. 75–79, 2005, ISSN: 1555-8584.
An anti-Tat hairpin ribozyme and a TAR RNA decoy were combined in one molecule. The chimeric molecule strongly inhibited HIV-1 replication (measured as changes in p24 levels in viral replication assays). The inhibitory action of the ribodecozyme (85%) was significantly greater than that shown by ribozyme and a non-catalytic variant carrying the functional decoy RNA domain (55% and 35%, respectively). This represents a significant improvement of the inhibitory efficiency of the ribozyme, suggesting there is an additive inhibitory effect on HIV-1 replication by the catalytic and decoy domains. This strategy could be used to create new inhibitor RNAs with enhanced in vivo performance.
- Berzal-Herranz A, Briones C, Sanchez-Luque FJ, Manrubia SC, Stich M. Molecules Inhibiting the Human Immunodeficiency Virus Type 1 (HIV-1), Method for the Production Thereof and Applications of Same. Jan 21st, 2016 (Filled in Nov 22nd, 2013). Publication N0. Europe 13857023.9 – 1401 PCT/ES2013070809.
- Sanchez-Luque FJ. RNAs inhibidores frente al virus de la inmunodeficiencia humana. Universidad de Granada. Jan 1st, 2013. Supervisor: Berzal-Herranz, A. Dr.
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