Inosine-Containing dsRNA Binds a Stress-Granule-like Complex and Downregulates Gene Expression In trans

Summary Long double-stranded RNAs (dsRNAs) may undergo extensive modification (hyperediting) by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine (A) residues are changed to inosine (I). Traditionally, consequences of A-to-I editing were thought to be limited to modified RNA itself. We show here, however, that hyperedited dsRNA (I-dsRNA) is able to downregulate gene expression in trans. Furthermore, we show that both endogenous expression and reporter gene expression were substantially reduced in the presence of I-dsRNA. This was due to a reduction in reporter mRNA levels and also translation inhibition. Importantly, we show that I-dsRNA interferes with translation initiation. We also show that I-dsRNA specifically binds a stress-granule-like complex. Stress granules (SGs) are important for translational silencing during stress. Finally, we propose a model whereby editing by ADARs results in downregulation of gene expression via SG formation.

Visualization was by enhanced chemiluminescence.

Immunoprecipitations (IPs)
10 µl of beads (Protein A Sepharose coupled to polyclonal α-TSN or PI serum) were mixed with 50 µl RNase A treated HeLa lysate in 1 ml NET buffer, and incubated with rotation for 2 h at 4 °C. Beads were washed extensively with NET buffer and proteins eluted using SDS sample buffer.
Antisera were affinity purified (Protein A Sepharose). 'AB' was equivalent to the antibody buffer.

In Vitro Translation with rTSN
In vitro translation was carried out essentially as described in experimental procedures. However, when 5 pmol rTSN (Scadden, 2005) was added to translations reactions, HeLa S100 was omitted. Translations with rTSN (or a control with HeLa S100) were carried out in the presence of 1.5 µM C or C-IU dsRNA. Luciferase activity is shown as a ratio of that seen with C-IU dsRNA relative to C dsRNA.

Figure S2. A "Stress-Protein Complex" Forms Specifically on I-dsRNA
(A) An RNA-protein complex was seen using EMSA when both IU and GU dsRNAs were incubated in HeLa cell lysate (lanes 2-4 and 5-7, respectively). This was not seen with buffer alone (lanes 1). While a dsRNA-protein complex formed with both IU and GU dsRNA, formation was less efficient with GU dsRNA. In addition to the RNA-protein complex, cleaved I-dsRNA was also readily detectable (lanes 2-4). A non-specific cleavage product was also seen (*). Addition of α-TSN resulted in a weak super-shifted species for both IU and GU dsRNAs (compare lanes 3 and 6, respectively, with lanes 2, 4, 5 and 7), whose mobility was only slightly less than the shifted band. A longer exposure of the top part of the gel is shown. As the super-shift was relatively difficult to visualize, the presence of TSN in the complex was additionally verified by EMSA using a HeLa extract where TSN was depleted using RNAi (data not shown).

Figure S3. TSN Is Required for Translation Inhibition by I-dsRNA
(A) An immunoblot of HeLa S100 ('H') and RRL was probed with various antibodies against SG components.
(B) Pp-luc mRNA was translated using RRL alone or RRL supplemented with either rTSN or HeLa S100, in the presence of 1.5 µM C or C-IU dsRNA (n=6). Translation was subsequently measured using luciferase assays, and was expressed as a ratio of luciferase activity in the presence of C-IU relative to C. All error bars are mean ± SD, n ≥ 3. A Student's t-test (2-tails, unequal variance) gave a P-value of <0.005.

Upf1
Regulator of nonsense transcripts (RENT1) 4 The I-dsRNA binding proteins identified are shown, and the number of unique peptides obtained for each protein is given. Previous characterization as a SG component is indicated for each protein.