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Novel insight into the non-coding repertoire through deep sequencing analysis.

Isakov O, Ronen R, Kovarsky J, Gabay A, Gan I, Modai S, Shomron N - Nucleic Acids Res. (2012)

Bottom Line: This diverse family of untranslated RNA molecules play a crucial role in cellular function.Using RandA, we reveal the complexity of the ncRNA repertoire in a given cell population.We further demonstrate the relevance of such an extensive ncRNA analysis by elucidating a multitude of characterizing features in pathogen infected mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
Non-coding RNAs (ncRNA) account for a large portion of the transcribed genomic output. This diverse family of untranslated RNA molecules play a crucial role in cellular function. The use of 'deep sequencing' technology (also known as 'next generation sequencing') to infer transcript expression levels in general, and ncRNA specifically, is becoming increasingly common in molecular and clinical laboratories. We developed a software termed 'RandA' (which stands for ncRNA Read-and-Analyze) that performs comprehensive ncRNA profiling and differential expression analysis on deep sequencing generated data through a graphical user interface running on a local personal computer. Using RandA, we reveal the complexity of the ncRNA repertoire in a given cell population. We further demonstrate the relevance of such an extensive ncRNA analysis by elucidating a multitude of characterizing features in pathogen infected mammalian cells. RandA is available for download at http://ibis.tau.ac.il/RandA.

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ncRNA transcript expression in the uninfected versus the infected samples (Base mean 1 and Base mean 2, respectively). This figure demonstrates the reduction of miRNA expression (red) and the induction of splicosomal RNA expression (blue) when inspecting the significantly different transcripts (non-gray; P < 0.01).
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gks228-F3: ncRNA transcript expression in the uninfected versus the infected samples (Base mean 1 and Base mean 2, respectively). This figure demonstrates the reduction of miRNA expression (red) and the induction of splicosomal RNA expression (blue) when inspecting the significantly different transcripts (non-gray; P < 0.01).

Mentions: Running RandA with a defined database which includes viral transcript sequences did not result in any HIV-related ncRNA transcripts (data not shown), despite its presence in the samples. This might be due to the scarcity of HIV-related sequences in the sample tested or the shortage of these in the Rfam database. Yet, we asked whether the profile of the human ncRNA transcripts in the infected sample can demonstrate features that strongly support an HIV infection (Figure 2). When focusing only on the miRNA transcripts in both samples, we noticed a substantial decrease in expression in a large proportion of miRNAs (96%) demonstrating a significant down-regulation in miRNA levels compared to other ncRNAs (Fisher's exact test; P < 0.0001; Figure 3). This was confirmed by real time PCR on the six most significantly differentially expressed miRNAs (P < 0.0001) that are found in both Rfam and in the commercial real time PCR array used (Table 2). The decreased miRNA expression in HIV infected human cells coincides with previously reported studies (30–32) and can be attributed to the suspected Dicer-suppressive effect exerted by HIV-1 Tat protein and/or TAR RNA (33). We further examined the most significantly decreased miRNAs [using DIANA-mirPath (34)] and observed a noteworthy enrichment (P < 0.001) of miRNA-targeted genes in the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway modulates and induces HIV infectivity (35–37). Thus, we speculate that this decrease in MAPK pathway genes-targeting miRNAs could serve as a viral mechanism to induce pathway activity and subsequent increased infectivity. This requires further experimental validation. MiRNA expression was not the only ncRNA group that changed after infection. We identified an enrichment of spliceosomal RNAs in the infected versus non-infected samples (Fisher's exact test; P < 0.01; Figure 3). Since HIV is known to disrupt the process of splicosome assembly in the nucleus (38,39), enrichment of such spliceosomal RNA fragments might support the presence of HIV in the infected samples. This could be a direct spliceosomal outcome or via changing the stability of splicing factors. Again, further experimentation is required for validation. Finally, 7SK RNA demonstrated a significant increase in expression in the infected sample (fold change >200; P < 4 ×e−22), suggesting a cellular antiviral defense mechanism given its reported disruption of HIV transcription (40).Figure 2.


Novel insight into the non-coding repertoire through deep sequencing analysis.

Isakov O, Ronen R, Kovarsky J, Gabay A, Gan I, Modai S, Shomron N - Nucleic Acids Res. (2012)

ncRNA transcript expression in the uninfected versus the infected samples (Base mean 1 and Base mean 2, respectively). This figure demonstrates the reduction of miRNA expression (red) and the induction of splicosomal RNA expression (blue) when inspecting the significantly different transcripts (non-gray; P < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367215&req=5

gks228-F3: ncRNA transcript expression in the uninfected versus the infected samples (Base mean 1 and Base mean 2, respectively). This figure demonstrates the reduction of miRNA expression (red) and the induction of splicosomal RNA expression (blue) when inspecting the significantly different transcripts (non-gray; P < 0.01).
Mentions: Running RandA with a defined database which includes viral transcript sequences did not result in any HIV-related ncRNA transcripts (data not shown), despite its presence in the samples. This might be due to the scarcity of HIV-related sequences in the sample tested or the shortage of these in the Rfam database. Yet, we asked whether the profile of the human ncRNA transcripts in the infected sample can demonstrate features that strongly support an HIV infection (Figure 2). When focusing only on the miRNA transcripts in both samples, we noticed a substantial decrease in expression in a large proportion of miRNAs (96%) demonstrating a significant down-regulation in miRNA levels compared to other ncRNAs (Fisher's exact test; P < 0.0001; Figure 3). This was confirmed by real time PCR on the six most significantly differentially expressed miRNAs (P < 0.0001) that are found in both Rfam and in the commercial real time PCR array used (Table 2). The decreased miRNA expression in HIV infected human cells coincides with previously reported studies (30–32) and can be attributed to the suspected Dicer-suppressive effect exerted by HIV-1 Tat protein and/or TAR RNA (33). We further examined the most significantly decreased miRNAs [using DIANA-mirPath (34)] and observed a noteworthy enrichment (P < 0.001) of miRNA-targeted genes in the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway modulates and induces HIV infectivity (35–37). Thus, we speculate that this decrease in MAPK pathway genes-targeting miRNAs could serve as a viral mechanism to induce pathway activity and subsequent increased infectivity. This requires further experimental validation. MiRNA expression was not the only ncRNA group that changed after infection. We identified an enrichment of spliceosomal RNAs in the infected versus non-infected samples (Fisher's exact test; P < 0.01; Figure 3). Since HIV is known to disrupt the process of splicosome assembly in the nucleus (38,39), enrichment of such spliceosomal RNA fragments might support the presence of HIV in the infected samples. This could be a direct spliceosomal outcome or via changing the stability of splicing factors. Again, further experimentation is required for validation. Finally, 7SK RNA demonstrated a significant increase in expression in the infected sample (fold change >200; P < 4 ×e−22), suggesting a cellular antiviral defense mechanism given its reported disruption of HIV transcription (40).Figure 2.

Bottom Line: This diverse family of untranslated RNA molecules play a crucial role in cellular function.Using RandA, we reveal the complexity of the ncRNA repertoire in a given cell population.We further demonstrate the relevance of such an extensive ncRNA analysis by elucidating a multitude of characterizing features in pathogen infected mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
Non-coding RNAs (ncRNA) account for a large portion of the transcribed genomic output. This diverse family of untranslated RNA molecules play a crucial role in cellular function. The use of 'deep sequencing' technology (also known as 'next generation sequencing') to infer transcript expression levels in general, and ncRNA specifically, is becoming increasingly common in molecular and clinical laboratories. We developed a software termed 'RandA' (which stands for ncRNA Read-and-Analyze) that performs comprehensive ncRNA profiling and differential expression analysis on deep sequencing generated data through a graphical user interface running on a local personal computer. Using RandA, we reveal the complexity of the ncRNA repertoire in a given cell population. We further demonstrate the relevance of such an extensive ncRNA analysis by elucidating a multitude of characterizing features in pathogen infected mammalian cells. RandA is available for download at http://ibis.tau.ac.il/RandA.

Show MeSH
Related in: MedlinePlus