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Papers by Daniel Barajas
Methods in Molecular Biology, 2011
microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate m... more microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs generates 3' end cleavage products that contain -ligation-competent, 5'-monophosphates. Precise mapping of miRNA-directed cleavage sites within target transcripts is, therefore, possible using RNA ligase-mediated 5' amplification of cDNA ends (RLM-RACE). Here, we provide a comprehensive RLM-RACE-based protocol for the amplification of 5' ends derived from cleaved transcripts resulting from miRNA-guided cleavage events. Novel strategies for high-throughput analysis of miRNA cleavage products have emerged as powerful tools for the novo identification of miRNA targets in a genomic perspective. In this work, we also describe a novel methodology for genome-wide identification of miRNA targets that exploits RLM-RACE for non-sequence-specific enrichment of cleaved transcripts, T7 RNA polymerase-mediated amplification of target products, and microarray hybridization.
Virology, 2008
El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido ... more El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido facilitado todavía por el investigador a cargo del archivo del mismo.
Journal of Virology, 2008
In plants, small RNA-guided processes referred to as RNA silencing control gene expression and se... more In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout t...
Virology, 2012
To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, ... more To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92 pol was reduced when Wwm1 was over-expressed, suggesting that the stability of p92 pol might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy.
Methods in Molecular Biology, 2011
microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate m... more microRNAs (miRNAs) regulate gene expression through sequence-specific interactions with cognate mRNAs that result in translational inhibition, mRNA decay, or slicing within the region of complementarity. miRNA processing activity on complementary target mRNAs generates 3' end cleavage products that contain -ligation-competent, 5'-monophosphates. Precise mapping of miRNA-directed cleavage sites within target transcripts is, therefore, possible using RNA ligase-mediated 5' amplification of cDNA ends (RLM-RACE). Here, we provide a comprehensive RLM-RACE-based protocol for the amplification of 5' ends derived from cleaved transcripts resulting from miRNA-guided cleavage events. Novel strategies for high-throughput analysis of miRNA cleavage products have emerged as powerful tools for the novo identification of miRNA targets in a genomic perspective. In this work, we also describe a novel methodology for genome-wide identification of miRNA targets that exploits RLM-RACE for non-sequence-specific enrichment of cleaved transcripts, T7 RNA polymerase-mediated amplification of target products, and microarray hybridization.
Virology, 2008
El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido ... more El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido facilitado todavía por el investigador a cargo del archivo del mismo.
Journal of Virology, 2008
In plants, small RNA-guided processes referred to as RNA silencing control gene expression and se... more In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout t...
Virology, 2012
To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, ... more To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92 pol was reduced when Wwm1 was over-expressed, suggesting that the stability of p92 pol might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy.