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.
Highlights
The conventional and recently identified novel functional roles of RNA underscore their continuing emerging importance
These observations point to the possibility of additional capped sRNA biotypes and to additional kinds of chemical modifications associated with the cap structures
Detection of capped sRNA with CAP Trapper To detect the presence of caps at the 59 end of sRNAs, we prepared sequence libraries using three different but complementary procedures (Figure 1)
Summary
The conventional and recently identified novel functional roles of RNA underscore their continuing emerging importance. A cause for this dearth of knowledge lies primarily in the reasoning that sRNAs cannot be mapped as accurately as long RNAs as well as that these sRNAs are random short-lived degradation products of long RNAs and are biologically unimportant These views have more recently come under reconsideration with the identification of novel biotypes of biologically active sRNAs. For example, sRNAs derived from processed snoRNAs [12] and tRNAs [8,13] have been identified as being involved in gene regulation and cell proliferation, respectively. Other capped sRNAs and a cytosolic capping protein complexes have been identified during studies involving the cleavage and recapping of long RNAs [14,19] These observations point to the possibility of additional capped sRNA biotypes and to additional kinds of chemical modifications associated with the cap structures. We have applied pre[4,20,21] and post-genomics methods to sRNAs extracted from the THP-1 cell line to explore the range of chemical modifications found associated with the 59cap structures
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