Multiplicity of 5′ Cap Structures Present on Short RNAs

<div><p>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 <i>type 0</i> 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.</p></div

Summary of cap structures detected in liquid chromatography-fractionated sRNA.

<p>Abbreviations: ND, not detected; Nm, ribose methylated ribonucleic acid; mN, base monomethylated ribonucleic acid; 2 mN, base dimethylated ribonucleic acid; 3 mN, base trimethylated ribonucleic acid; Yn, unknown structure 2mGpppAm(1) and 2mGpppAm(2) are isomer with different retention time on LC-MS, which may be 2,2mGpppAm and 2,7mGpppAm. Note that the order of the 5′ end nucleotide is not predictable by mass spectroscopy.</p

Thin layer chromatography reveals cap structures on sRNAs at all length ranges.

<p>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.</p

Differential expression analysis of annotated sRNAs.

<p>Enrichment of annotated tag clusters in the <i>IP K121</i> and <i>Capped sRNA</i> 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.</p

Construction of sequence libraries enriched for capped sRNAs.

<p>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.</p

Comparison of libraries identifies enriched sRNA lengths and annotations.

<p>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. <i>None</i> means that no annotation was found, and <i>other</i> combines all annotations categories that were not listed individually.</p

2,2,7 mGp is the most strongly detected methylguanosine in all length ranges.

<p>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.</p

Summary of cap structures that were cross-validated by 2D-TLC and mass spectroscopy.

<p>*: Unique to this fraction.</p><p>**: First time to be reported in mammals.</p

The monocyte core TRN elements.

<p>Rfc = differential expression fold change rank.</p><p>Rco = co-occurrence rank.</p><p>IS = Importance Score (1/Rfc+1/Rco).</p

Four TRN-inducers adopted inflammatory cytokine secretion in response to LPS.

<p>FIB-mock and FIB-4Fs were treated or untreated with 10 µg/ml LPS. The cytokine levels of supernatant medium were assessed using the Proteome Profiler Human Cytokine Array, Pannel A. Array images were collected by LAS-3000 imaging system (A). The intensity of each spot was determined by Multi Gauge softwere (B).</p