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. 2014 Jul 31;9(7):e102895.
doi: 10.1371/journal.pone.0102895. eCollection 2014.

Multiplicity of 5' cap structures present on short RNAs

Rehab F Abdelhamid  1 , Charles Plessy  1 , Yoshio Yamauchi  2 , Masato Taoka  2 , Michiel de Hoon  1 , Thomas R Gingeras  3 , Toshiaki Isobe  2 , Piero Carninci  1
Affiliations

Multiplicity of 5' cap structures present on short RNAs

Rehab F Abdelhamid et al. PLoS One. .

Abstract

Most RNA molecules are co- or post-transcriptionally modified to alter their chemical and functional properties to assist in their ultimate biological function. Among these modifications, the addition of 5' cap structure has been found to regulate turnover and localization. Here we report a study of the cap structure of human short (<200 nt) RNAs (sRNAs), using sequencing of cDNA libraries prepared by enzymatic pretreatment of the sRNAs with cap sensitive-specificity, thin layer chromatographic (TLC) analyses of isolated cap structures and mass spectrometric analyses for validation of TLC analyses. Processed versions of snoRNAs and tRNAs sequences of less than 50 nt were observed in capped sRNA libraries, indicating additional processing and recapping of these annotated sRNAs biotypes. We report for the first time 2,7 dimethylguanosine in human sRNAs cap structures and surprisingly we find multiple type 0 cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in RNA length fractions shorter than 50 nt. Finally, we find the presence of additional uncharacterized cap structures that wait determination by the creation of needed reference compounds to be used in TLC analyses. These studies suggest the existence of novel biochemical pathways leading to the processing of primary and sRNAs and the modifications of their RNA 5' ends with a spectrum of chemical modifications.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Construction of sequence libraries enriched for capped sRNAs.
Step-by-step graphical summary of the construction of the sRNA libraries. The bottom line indicates the library name; in the manuscript, these names are emphasized in italics. The key methods are immunoprecipitation for the IP K121 and IP control libraries, TAP (or mock) treatment for the Capped sRNA and sRNA libraries, and CAP Trapper for the sRNA CAGE libraries.
Figure 2
Figure 2. Comparison of libraries identifies enriched sRNA lengths and annotations.
A–C: Size profile of the immunoprecipitated (A), TAP-treated (B) and CAP trapped sRNA libraries and their controls. D: Annotations per length range, represented by stacked bar plots for each library replicate. None means that no annotation was found, and other combines all annotations categories that were not listed individually.
Figure 3
Figure 3. Differential expression analysis of annotated sRNAs.
Enrichment of annotated tag clusters in the IP K121 and Capped sRNA libraries respectively to their controls. Horizontal and vertical axis: fold changes in logarithmic scale. Each dot represents one cluster, with a size proportional to its average expression.
Figure 4
Figure 4. Thin layer chromatography reveals cap structures on sRNAs at all length ranges.
A–F: 2D-TLC of radiolabelled cap structures. Long cleavage products corresponding to sRNAs remained immobile on the spotting origin (bottom left corners), and dinucleotides formed large spots at the lower right corner and mid left side, while the cap structures and mononucleotide degradation products migrated further up and right. The first dimension was run using solvent A (see materials and methods) and is displayed from bottom to the top. The second dimension was run using solvent B (left to right). Three size fractions were investigated: <50 nt (A–B), 50–100 nt (C–D), and 100–200 nt (E–F). The position of cold mononucleotides Ap, Gp, Cp, Up detected by UV shadowing is indicated by purple dashed ellipses. B, D, F: chromatographies in presence of radiolabelled standards for Gp, 7 mGp, 2,7 mGp and 2,2,7 mGp. Spots were assigned according to the reference Kieth 1995 or referred to as X.
Figure 5
Figure 5. 2,2,7 mGp is the most strongly detected methylguanosine in all length ranges.
Quantitative analysis of the 2D-TLC, normalized as a percentage of total intensity for each RNA fraction. Error bars represent the standard deviation on three replicates.
See this image and copyright information in PMC

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