Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3'UTRs

Abstract

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3' untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3'UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3'UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3'UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5'-3' exonucleolytic (XRN1) function in knockdown experiments in human cells.

Keywords: RNA stability element; Rous sarcoma virus; deadenylation; decapping; long 3′UTR; nonsense mediated decay.

Figure 1

(A) Schematic of the Rous sarcoma virus (RSV) unspliced mRNA. The 400-nt RNA stability element (RSE) (shown in green) is located immediately downstream of the gag stop codon. This RNA has a very long 3′ untranslated region (UTR) of ca. 7 kb. (B) Schematic of tet-regulated β-globin reporter mRNA constructs used in our studies. WT-SMG5 mRNA contains the β-globin sequence followed by the SMG5 3′UTR sequence. The RSV RSE sequence was inserted into this reporter construct immediately after the β-globin stop codon to generate RSE-SMG5.

Figure 2

RNA stability element inhibits deadenylation. (A) Agarose northern blot showing deadenylation and decay of WT-SMG5 and RSE-SMG5 mRNAs following tet-off transcriptional pulse chase. RNA was subjected to oligonucleotide-directed RNaseH treatment prior to Northern blotting for tail length determination. Lanes marked “dT” show the length of the RNA fragment without a poly(A) tail; 1 kb plus DNA ladder (ThermoFisher Catalog number: 10787018, Halethorpe, MD, USA) was end labelled and used for size determination (size corrected for RNA). Wildtype β-globin (βwt) (upper band) was used as a loading control. (B) Poly(A) tail length determined from the gel was plotted against time to show the initial and final tail length of the two reporter constructs. The plotted values are the average of at least three separate experiments. Error bars show the standard error. Deadenylation rate of the reporters were also determined from this analysis. (C) Deadenylation and decay of the reporter mRNAs were compared by plotting mRNA remaining (% of original) and poly(A) length remaining (% of original tail length) on the same plot.

Figure 3

Depletion of decapping mRNA 2 (DCP2) or 5′-3′ Exoribonuclease 1(XRN1) leads to the accumulation of reporter mRNAs. (A) mRNA levels of the two reporters isolated from cells depleted for Exoribonuclease 1 (XRN1 KD), decapping mRNA 2 (DCP2 KD), or scrambled control (scr) were detected by agarose Northern blotting. NeoR, an mRNA produced from the same plasmid that generates the tet-off β-globin constructs was used as a loading control. Mean numbers show the amount of mRNA relative to scrambled control for WT-SMG5. SE shows the standard error of the values. (B) Left panel shows levels of WT-SMG5 reporter in different knockdown conditions normalized to the scrambled control. Right panel shows levels of RSE-SMG5 reporter in different knockdown conditions normalized to the corresponding scrambled control. The plotted values are the average of at least three separate experiments. Error bars show the standard error. (C) Quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the levels of knockdown of DCP2 and XRN1. Expression levels of these mRNAs were measured relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Unpaired t-test was used to test for significance of the change in expression between each knockdown and the corresponding scrambled control. * denotes p-value < 0.05. ** p-value < 0.01.