Hidden layers of human small RNAs - PubMed (original) (raw)
Comparative Study
doi: 10.1186/1471-2164-9-157.
Mari Nakamura, Yukari Takahashi, Albin Sandelin, Shintaro Katayama, Shiro Fukuda, Carsten O Daub, Chikatoshi Kai, Jun Kawai, Jun Yasuda, Piero Carninci, Yoshihide Hayashizaki
Affiliations
- PMID: 18402656
- PMCID: PMC2359750
- DOI: 10.1186/1471-2164-9-157
Comparative Study
Hidden layers of human small RNAs
Hideya Kawaji et al. BMC Genomics. 2008.
Abstract
Background: Small RNA attracts increasing interest based on the discovery of RNA silencing and the rapid progress of our understanding of these phenomena. Although recent studies suggest the possible existence of yet undiscovered types of small RNAs in higher organisms, many studies to profile small RNA have focused on miRNA and/or siRNA rather than on the exploration of additional classes of RNAs.
Results: Here, we explored human small RNAs by unbiased sequencing of RNAs with sizes of 19-40 nt. We provide substantial evidences for the existence of independent classes of small RNAs. Our data shows that well-characterized non-coding RNA, such as tRNA, snoRNA, and snRNA are cleaved at sites specific to the class of ncRNA. In particular, tRNA cleavage is regulated depending on tRNA type and tissue expression. We also found small RNAs mapped to genomic regions that are transcribed in both directions by bidirectional promoters, indicating that the small RNAs are a product of dsRNA formation and their subsequent cleavage. Their partial similarity with ribosomal RNAs (rRNAs) suggests unrevealed functions of ribosomal DNA or interstitial rRNA. Further examination revealed six novel miRNAs.
Conclusion: Our results underscore the complexity of the small RNA world and the biogenesis of small RNAs.
Figures
Figure 1
Classification flow of small RNAs. Some sequences are alignable with several classes of RNAs and multiple loci on the genome. We assign one sequence to one class based on this flow.
Figure 2
Content and conservation properties of each sequenced fraction. a) The count of small RNAs mapping to specific types of annotation is shown as histograms for the short and long libraries. Classifications are based on the process described in Fig 2 and Methods. b) The mean PhastCons [21] scores for the region -300 to +300 relative to the midpoint of each small RNA cluster are shown, broken up into two fractions. Position 0 indicates the midpoint of each cluster. For comparison, 500 equally sized randomly selected regions where the midpoint was either exonic, intronic, or intergenic was analyzed in the same way. The small fraction is generally highly conserved, which is expected due to many potential miRNA sequences it contains. The longer fraction is on average less conserved than exonic sequences, but considerably higher conserved than intergenic or intronic regions.
Figure 3
Length distributions of small RNAs based on our classifications. Note that the bimodal length distributions of the total small RNAs can be due to our library construction protocol, where shorter and longer fractions are prepared separately. The length distribution per each RNA class demonstrates distinct preferences between these classes.
Figure 4
Small RNAs assigned to tRNA. (A) An alignment of small RNAs assigned to HisGTG tRNA. Only the top six sequences are aligned with the complete sequence of His tRNA. (B) – (E) Schematic representation of tRNA cloverleaf structures, which are obtained from the Genomic tRNA database [32], and aligned regions of major small RNAs assigned to the tRNA. Bold font indicates small RNA sequences. Gray color indicates added "CCA" in tRNA maturation process, which is not encoded in the genome.
Figure 5
Northern blotting of tRNA fragments for some tissues and cell lines. Arrows show the bands corresponding to the small RNA sequences assigned to tRNAs. See the Methods section for the detail of primer sequence and RNA samples.
Figure 6
Schematic representation of genomic regions, where the small RNAs assigned to known genes are mapped on. Clusters of the small RNAs are represented in blue, transcription starts captured by CAGE are represented in pink (supported with single CAGE tag) and red (supported by two or more tags) [45]. Antisense transcription to the gene, which is near to the small RNAs, is represented as arrow on the genome representation.
Figure 7
Example of small RNAs overlapping a bidirectional promoter. The histograms show frequencies of transcription start sites (TSS) captured by CAGE. Top and bottom panels show CAGE tag-defined TSS mapping to forward and reverse strand, respectively. The shaded grey box indicates the region with potential for forming a dsRNA by transcription on both strands. The location of the small RNA cluster inside this region is indicated in the central panel as a white box. The numbering of genomic nucleotides is assigned by defining the center of the small RNA cluster as +1.
References
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