Expression of distinct RNAs from 3' untranslated regions - PubMed (original) (raw)

. 2011 Mar;39(6):2393-403.

doi: 10.1093/nar/gkq1158. Epub 2010 Nov 12.

Dagmar Wilhelm, Marcel E Dinger, Giulia Soldà, Darren J Korbie, Evgeny A Glazov, Vy Truong, Maren Schwenke, Cas Simons, Klaus I Matthaei, Robert Saint, Peter Koopman, John S Mattick

Affiliations

Expression of distinct RNAs from 3' untranslated regions

Tim R Mercer et al. Nucleic Acids Res. 2011 Mar.

Abstract

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.

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Figures

Figure 1.

Figure 1.

Post-transcriptional processing of 3′UTRs. (A) Full-length cDNA transcripts associated with 3′UTRs. Histogram (left) shows enrichment of CAGE tags with the 5′ termini of cDNAs mapping within 3′UTRs (i.e. uaRNAs). Histogram (right) shows correlated enrichment of the terminal regions of these cDNA transcripts with the 3′ end of the host RefSeq gene. (B) The top panel shows a schematic representation of the mapping strategy employed to discern CAGE tags that span exon–exon junctions (EEJs) and therefore suggest post-transcriptional processing. The lower panels show the distribution of CAGE tags mapping proximal to EEJs in human (left) and mouse (right). The frequencies of CAGE tags that map uniquely to the genome (blue) are under-represented adjacent to EEJs. This under-representation is reconciled by considering CAGE tags that map across EEJs (red).

Figure 2.

Figure 2.

Specific and distinct expression profiles for 3′UTRs in mouse tissues. (A) Cluster analysis of the expression of the 500 genes containing the highest 3′UTR CAGE tag frequency shows the differential expression levels (no expression, black; high expression, red) of 3′UTRs in various mouse tissues. Expression level was determined as the normalized CAGE tag frequency mapping to 3′UTRs. (B) The diverse ratios of promoter to 3′UTR CAGE frequency (high, green; low, red) for each tissue indicates independent expression of mRNAs and 3′UTRs. (C) Illustrative examples indicating promoter (blue) and 3′UTR (red) CAGE frequency. tpm, tags per million; Li, liver; E, embryo; Lu, lung; M, macrophage; C, cerebellum; H, hippocampus; VC, visual cortex; SC, somatic cortex.

Figure 3.

Figure 3.

Illustrative examples of 3′UTR transcripts in mouse and fruit fly. (A) Schematic representation of CAGE tag clusters in the 3′UTR of the mouse Camk2a gene from somatosensory cortex (green), visual cortex (orange) and hippocampus (blue). Different sites with high frequency of CAGE tags map preferentially in hippocampus (68 tags per million; tpm), the visual cortex (52 tpm) and somato-sensory cortex (56 tpm). (B) Schematic representation of the fruit fly gene oskar showing specific SAGE tags in the 3′UTR in young and old females.

Figure 4.

Figure 4.

uaRNAs within Col1a1 3′UTR. (A) Genome browser view of Col1a1 3′UTR showing; histogram of CAGE distribution and density (top panel; tpm, tags per million), 5′ ends inferred from high confidence 5′ RACE products (second panel, black bars); riboprobes used in in situ hybridization (third panel; green bars); Col1a1 annotated coding sequence (CDS; black bar) and 3′UTR (blue bar); reverse transcriptase (RT, 1–3) and PCR primers (A and B) used in (B) (black arrows). (B) Confirmation of 3′UTR annotation by RT using primers B, 1, 2 or 3 followed by PCR using primers A (forward) and B (reverse). Lanes contain (from left to right) 1-kb plus ladder (M), positive CDS control primer (B), RT primers (1–3) and no RT negative control (-ve). (C) ISH using one riboprobe in the terminal constitutive coding exon (CDS, dark green) of the Col1a1 gene and two probes (uaRNA1 and uaRNA2, light green) corresponding to 3′UTR sequences, one downstream of each of the two 5′ RACE clusters. All three probes exhibit expression at sites of chondrogenesis, such as the otic vesicle (upper panel, black arrowheads) and the developing ribs (lower panel, red arrowheads). Expression of the coding region is apparent during ossification of vertebrae, in contrast to both uaRNAs whose expression is absent (lower panel, green arrowheads). The uaRNA2 probe detects expression in the the dorsal root ganglia (lower panel, green asterisks); this expression is not detected by the CDS or uaRNA1 probes. Higher magnification of the dorsal root ganglia shows that the uaRNA expression is localized to the nucleus (lower panel, green asterisk).

Figure 5.

Figure 5.

uaRNAs within the Nfia and Myadm 3′UTRs. (A) (Top panel) Genomic context of Nfia 3′UTR showing annotated coding sequence (CDS, black) and 3′UTR (blue), histogram of CAGE distribution and density (tpm, tags per million), and riboprobes used in ISH that target the terminal constitutive coding exons (dark green; CDS) and 3′UTR (light green; uaRNA1). (Bottom panel) Section ISH showing the Nfia CDS probe detecting expression in interstitial cells (green asterisks) of 12.5 dpc testes (marker) and the uaRNA probe detecting expression within the testis cords (red asterisks). (B) (Top panel) Genomic context of Myadm 3′UTR showing annotated coding sequence (CDS, black) and 3′UTR (blue), riboprobes used in ISH that target the terminal constitutive coding exon (dark green; CDS) and 3′UTR (light green; uaRNA1). (Bottom panel) Section ISH showing the Myadm CDS probe detecting expression in the cytoplasm of interstitial cells (green asterisks) in the developing testis at 12.5 dpc and the uaRNA probe detecting expression in the nuclei of Sertoli cells and germ cells in the testis cords (red asterisks). High Myadm expression was not detected by either the CDS or the uaRNA probe elsewhere in the embryo (see

Supplementary Figure S8

).

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