Limits...
Global analysis of yeast RNA processing identifies new targets of RNase III and uncovers a link between tRNA 5' end processing and tRNA splicing.

Hiley SL, Babak T, Hughes TR - Nucleic Acids Res. (2005)

Bottom Line: We also monitored the accumulation of improperly processed flank sequences of pre-RNAs in strains depleted for known RNA nucleases, including RNase III, Dbr1p, Xrn1p, Rat1p and components of the exosome and RNase P complexes.Among the hundreds of aberrant RNA processing events detected, two novel substrates of Rnt1p (the RUF1 and RUF3 snoRNAs) were identified.We also identified a relationship between tRNA 5' end processing and tRNA splicing, processes that were previously thought to be independent.

View Article: PubMed Central - PubMed

Affiliation: Banting and Best Department of Medical Research, University of Toronto 112 College Street, Toronto, ON M5G 1L6, Canada.

ABSTRACT
We used a microarray containing probes that tile all known yeast noncoding RNAs (ncRNAs) to investigate RNA biogenesis on a global scale. The microarray verified a general loss of Box C/D snoRNAs in the TetO7-BCD1 mutant, which had previously been shown for only a handful of snoRNAs. We also monitored the accumulation of improperly processed flank sequences of pre-RNAs in strains depleted for known RNA nucleases, including RNase III, Dbr1p, Xrn1p, Rat1p and components of the exosome and RNase P complexes. Among the hundreds of aberrant RNA processing events detected, two novel substrates of Rnt1p (the RUF1 and RUF3 snoRNAs) were identified. We also identified a relationship between tRNA 5' end processing and tRNA splicing, processes that were previously thought to be independent. This analysis demonstrates the applicability of microarray technology to the study of global analysis of ncRNA synthesis and provides an extensive directory of processing events mediated by yeast ncRNA processing enzymes.

Show MeSH

Related in: MedlinePlus

Longer oligonucleotide probes improve the sensitivity of detection. The intensity of fluorescence of wild-type RNA bound to probes on either short (top) or long (bottom) oligonucleotide microarrays, shaded according to the color bar on the right, is plotted for representative transcripts: (A) tRNAs, (B) 35S pre-rRNA and (C) snoRNAs. Schematic diagrams of the RNAs and flanking regions are shown below with boxes representing RNA sequence and thin lines indicating flanking sequence. (D) Box C/D RNA depletion in a TetO7-BCD1 strain. The relative fluorescence of Box C/D and Box H/ACA snoRNA probes is shown, colored according to the scale on the right. Note that because the reciprocal of the mutant:wild-type ratio is plotted in this panel, increasing red color indicates depletion of snoRNAs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1140755&req=5

fig1: Longer oligonucleotide probes improve the sensitivity of detection. The intensity of fluorescence of wild-type RNA bound to probes on either short (top) or long (bottom) oligonucleotide microarrays, shaded according to the color bar on the right, is plotted for representative transcripts: (A) tRNAs, (B) 35S pre-rRNA and (C) snoRNAs. Schematic diagrams of the RNAs and flanking regions are shown below with boxes representing RNA sequence and thin lines indicating flanking sequence. (D) Box C/D RNA depletion in a TetO7-BCD1 strain. The relative fluorescence of Box C/D and Box H/ACA snoRNA probes is shown, colored according to the scale on the right. Note that because the reciprocal of the mutant:wild-type ratio is plotted in this panel, increasing red color indicates depletion of snoRNAs.

Mentions: To increase the sensitivity of detection of directly labeled RNA transcripts, we created a version of our previously described microarray (11) with longer oligonucleotide probes (see Supplementary Material for probe sequences). This new microarray features probes tiled across the same ncRNA sequences, but has an average probe length of 25.1 nt [compared with 18.4 nt on the previous microarray (11)]. A comparison of the two microarrays using wild-type RNA revealed that the longer probes improved the consistency and detection levels of both tRNAs (Figure 1A) and rRNA (Figure 1B). Furthermore, we were able to detect a greater proportion of less abundant RNA transcripts (e.g. snoRNAs, Figure 1C). Although microarray spot intensity is a relative measurement (controlled by the photomultiplier tube voltage on the scanner), the fact that the flanking sequences (representing background) are slightly darker (i.e. there is lower signal-to-noise) in all of the short-oligonucleotide plots in Figure 1 confirms that the overall signal-to-noise ratio is superior on the long-oligonucleotide microarray.


Global analysis of yeast RNA processing identifies new targets of RNase III and uncovers a link between tRNA 5' end processing and tRNA splicing.

Hiley SL, Babak T, Hughes TR - Nucleic Acids Res. (2005)

Longer oligonucleotide probes improve the sensitivity of detection. The intensity of fluorescence of wild-type RNA bound to probes on either short (top) or long (bottom) oligonucleotide microarrays, shaded according to the color bar on the right, is plotted for representative transcripts: (A) tRNAs, (B) 35S pre-rRNA and (C) snoRNAs. Schematic diagrams of the RNAs and flanking regions are shown below with boxes representing RNA sequence and thin lines indicating flanking sequence. (D) Box C/D RNA depletion in a TetO7-BCD1 strain. The relative fluorescence of Box C/D and Box H/ACA snoRNA probes is shown, colored according to the scale on the right. Note that because the reciprocal of the mutant:wild-type ratio is plotted in this panel, increasing red color indicates depletion of snoRNAs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1140755&req=5

fig1: Longer oligonucleotide probes improve the sensitivity of detection. The intensity of fluorescence of wild-type RNA bound to probes on either short (top) or long (bottom) oligonucleotide microarrays, shaded according to the color bar on the right, is plotted for representative transcripts: (A) tRNAs, (B) 35S pre-rRNA and (C) snoRNAs. Schematic diagrams of the RNAs and flanking regions are shown below with boxes representing RNA sequence and thin lines indicating flanking sequence. (D) Box C/D RNA depletion in a TetO7-BCD1 strain. The relative fluorescence of Box C/D and Box H/ACA snoRNA probes is shown, colored according to the scale on the right. Note that because the reciprocal of the mutant:wild-type ratio is plotted in this panel, increasing red color indicates depletion of snoRNAs.
Mentions: To increase the sensitivity of detection of directly labeled RNA transcripts, we created a version of our previously described microarray (11) with longer oligonucleotide probes (see Supplementary Material for probe sequences). This new microarray features probes tiled across the same ncRNA sequences, but has an average probe length of 25.1 nt [compared with 18.4 nt on the previous microarray (11)]. A comparison of the two microarrays using wild-type RNA revealed that the longer probes improved the consistency and detection levels of both tRNAs (Figure 1A) and rRNA (Figure 1B). Furthermore, we were able to detect a greater proportion of less abundant RNA transcripts (e.g. snoRNAs, Figure 1C). Although microarray spot intensity is a relative measurement (controlled by the photomultiplier tube voltage on the scanner), the fact that the flanking sequences (representing background) are slightly darker (i.e. there is lower signal-to-noise) in all of the short-oligonucleotide plots in Figure 1 confirms that the overall signal-to-noise ratio is superior on the long-oligonucleotide microarray.

Bottom Line: We also monitored the accumulation of improperly processed flank sequences of pre-RNAs in strains depleted for known RNA nucleases, including RNase III, Dbr1p, Xrn1p, Rat1p and components of the exosome and RNase P complexes.Among the hundreds of aberrant RNA processing events detected, two novel substrates of Rnt1p (the RUF1 and RUF3 snoRNAs) were identified.We also identified a relationship between tRNA 5' end processing and tRNA splicing, processes that were previously thought to be independent.

View Article: PubMed Central - PubMed

Affiliation: Banting and Best Department of Medical Research, University of Toronto 112 College Street, Toronto, ON M5G 1L6, Canada.

ABSTRACT
We used a microarray containing probes that tile all known yeast noncoding RNAs (ncRNAs) to investigate RNA biogenesis on a global scale. The microarray verified a general loss of Box C/D snoRNAs in the TetO7-BCD1 mutant, which had previously been shown for only a handful of snoRNAs. We also monitored the accumulation of improperly processed flank sequences of pre-RNAs in strains depleted for known RNA nucleases, including RNase III, Dbr1p, Xrn1p, Rat1p and components of the exosome and RNase P complexes. Among the hundreds of aberrant RNA processing events detected, two novel substrates of Rnt1p (the RUF1 and RUF3 snoRNAs) were identified. We also identified a relationship between tRNA 5' end processing and tRNA splicing, processes that were previously thought to be independent. This analysis demonstrates the applicability of microarray technology to the study of global analysis of ncRNA synthesis and provides an extensive directory of processing events mediated by yeast ncRNA processing enzymes.

Show MeSH
Related in: MedlinePlus