Limits...
Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

Zywicki M, Bakowska-Zywicka K, Polacek N - Nucleic Acids Res. (2012)

Bottom Line: Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases.Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data.To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. marek.zywicki@i-med.ac.at

ABSTRACT
The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Show MeSH

Related in: MedlinePlus

Processing of a 23-mer from 5′-end of 25S ribosomal RNA. (A) The location of the detected processing product on the secondary structure diagram of large ribosomal subunit rRNA is depicted. (B) UCSC Genome Browser visualization of the APART tracks (green) within the region of contig loc.XII-464072_3929 containing the 23-mer. (C) Semi-quantitative RT–PCR with primers specific for the 23-mer using size-selected (10–50 nt) total RNA as template results in a 69-nt long PCR product. By using 10- to 50-nt long RNAs as template amplification of this 23-mer from the unprocessed full-length 25S rRNA is avoided.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351166&req=5

gks020-F4: Processing of a 23-mer from 5′-end of 25S ribosomal RNA. (A) The location of the detected processing product on the secondary structure diagram of large ribosomal subunit rRNA is depicted. (B) UCSC Genome Browser visualization of the APART tracks (green) within the region of contig loc.XII-464072_3929 containing the 23-mer. (C) Semi-quantitative RT–PCR with primers specific for the 23-mer using size-selected (10–50 nt) total RNA as template results in a 69-nt long PCR product. By using 10- to 50-nt long RNAs as template amplification of this 23-mer from the unprocessed full-length 25S rRNA is avoided.

Mentions: To substantiate these bioinformatically predicted RNA processing events, we have experimentally verified the presence of selected RNA fragments. For the most abundant class of ribosomal RNAs, we have selected a 23-nt long piece derived from the 5′-part of 25S rRNA found in 3901 copies that showed almost exactly identical ends (Figure 4). Experimental investigations confirmed the presence of this particular rRNA fragment under all investigated growth conditions (Figure 4C). Similar rRNA cleavage (however, primarily from the 3′-end of the 25S rRNA) has been already observed in S. cerevisiae during oxidative stress as well as during entry into stationary phase (39).Figure 4.


Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

Zywicki M, Bakowska-Zywicka K, Polacek N - Nucleic Acids Res. (2012)

Processing of a 23-mer from 5′-end of 25S ribosomal RNA. (A) The location of the detected processing product on the secondary structure diagram of large ribosomal subunit rRNA is depicted. (B) UCSC Genome Browser visualization of the APART tracks (green) within the region of contig loc.XII-464072_3929 containing the 23-mer. (C) Semi-quantitative RT–PCR with primers specific for the 23-mer using size-selected (10–50 nt) total RNA as template results in a 69-nt long PCR product. By using 10- to 50-nt long RNAs as template amplification of this 23-mer from the unprocessed full-length 25S rRNA is avoided.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351166&req=5

gks020-F4: Processing of a 23-mer from 5′-end of 25S ribosomal RNA. (A) The location of the detected processing product on the secondary structure diagram of large ribosomal subunit rRNA is depicted. (B) UCSC Genome Browser visualization of the APART tracks (green) within the region of contig loc.XII-464072_3929 containing the 23-mer. (C) Semi-quantitative RT–PCR with primers specific for the 23-mer using size-selected (10–50 nt) total RNA as template results in a 69-nt long PCR product. By using 10- to 50-nt long RNAs as template amplification of this 23-mer from the unprocessed full-length 25S rRNA is avoided.
Mentions: To substantiate these bioinformatically predicted RNA processing events, we have experimentally verified the presence of selected RNA fragments. For the most abundant class of ribosomal RNAs, we have selected a 23-nt long piece derived from the 5′-part of 25S rRNA found in 3901 copies that showed almost exactly identical ends (Figure 4). Experimental investigations confirmed the presence of this particular rRNA fragment under all investigated growth conditions (Figure 4C). Similar rRNA cleavage (however, primarily from the 3′-end of the 25S rRNA) has been already observed in S. cerevisiae during oxidative stress as well as during entry into stationary phase (39).Figure 4.

Bottom Line: Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases.Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data.To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. marek.zywicki@i-med.ac.at

ABSTRACT
The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Show MeSH
Related in: MedlinePlus