Translational control of the interferon regulatory factor 2 mRNA by IRES element

Translational control represents an important mode of regulation of gene expression under stress conditions. We have studied the translation of interferon regulatory factor 2 (IRF2) mRNA, a negative regulator of transcription of interferon-stimulated genes and demonstrated the presence of internal ribosome entry site (IRES) element in the 5'UTR of IRF2 RNA. Various control experiments ruled out the contribution of leaky scanning, cryptic promoter activity or RNA splicing in the internal initiation of IRF2 RNA. It seems IRF2-IRES function is not sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease. Interferon treatment did not affect the IRF2-IRES activity or the protein level significantly. Also, in cells treated with tunicamycin [an agent causing endoplasmic reticulum (ER) stress], the IRF2-IRES activity and the protein levels were unaffected, although the cap-dependent translation was severely impaired. Analysis of the cellular protein binding with the IRF2-IRES suggests certain cellular factors, which might influence its function under stress conditions. Interestingly, partial knockdown of PTB protein significantly inhibited the IRF2-IRES function. Taken together, it appears that IRF2 gene expression during stress condition is controlled by the IRES element, which in turn influences the cellular response

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

BACKGROUND: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. METHODOLOGY/PRINCIPAL FINDINGS: We have probed the putative secondary structure of the IRF2 5'UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3'terminus of the 5'untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2-5'UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3'end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA. CONCLUSION/SIGNIFICANCE: It appears that PTB binding to multiple sites within IRF2 5'UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation

Translational control of the interferon regulatory factor 2 mRNA by IRES element

Translational control represents an important mode of regulation of gene expression under stress conditions. We have studied the translation of interferon regulatory factor 2 (IRF2) mRNA, a negative regulator of transcription of interferon-stimulated genes and demonstrated the presence of internal ribosome entry site (IRES) element in the 5’UTR of IRF2 RNA. Various control experiments ruled out the contribution of leaky scanning, cryptic promoter activity or RNA splicing in the internal initiation of IRF2 RNA. It seems IRF2-IRES function is not sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease. Interferon a treatment did not affect the IRF2-IRES activity or the protein level significantly. Also, in cells treated with tunicamycin [an agent causing endoplasmic reticulum (ER) stress], the IRF2-IRES activity and the protein levels were unaffected, although the cap-dependent translation was severely impaired. Analysis of the cellular protein binding with the IRF2-IRES suggests certain cellular factors, which might influence its function under stress conditions. Interestingly, partial knockdown of PTB protein significantly inhibited the IRF2-IRES function. Taken together, it appears that IRF2 gene expression during stress condition is controlled by the IRES element, which in turn influences the cellular response

PTB protein helps interaction of other transacting factors with the IRF2-IRES:

<p>(A) ³²P labeled IRF2 WT 5′UTR and mIRF2 5′UTR were crosslinked with total HeLa S10 extracts and run on an 10% SDS PAGE followed by phosphorimaging. Lane 1 represents no protein control. Lane M indicates the molecular mass marker. (B) 70% confluent HeLa cells were transfected with 60 nm and 80 nm of siPTB or nsp si as indicated. 36 hrs post-transfection the cells were harvested and subjected for western blot analysis to check PTB levels. S10 extract was prepared from these cells and UV Crosslinking was performed as mentioned previously. Lane M indicates the molecular mass marker. (C) Western-blot analysis showing the reduced levels of PTB with increasing concentration of siPTB. Actin protein was detected as loading control.</p

Probing of secondary structure of IRF2 UTR RNA with DMS and RNase T1:

<p>(A) A schematic representation of IRF2-Luc monocistronic construct used for generating <i>in vitro</i> transcripts by using T7 RNA polymerase. A 20 nucleotide long -³²P labeled reverse primer was used for reverse transcription indicated by an arrow at the 5′ end of luciferase. (B) IRF2 5′ UTR RNA was incubated with (lane 2) or without (lane 1) dimethyl sulphate (DMS). The unmodified and modified RNAs were reverse transcribed and cDNAs were resolved parallel with a sequencing reaction performed with the same end labeled primer. Modified nucleotides are indicated on the right of the panel. (C) IRF2 5′ UTR RNA was incubated with or without of 1.0 unit of RNase T1 and the undigested and digested RNAs were reverse transcribed and cDNAs were resolved in parallel with a sequencing reaction performed with the same end labeled primer. Putative cleavage points are indicated by arrows on the right of the panel.</p

CD spectroscopic analysis of IRF2 in presence of PTB protein:

<p>CD spectra were obtained in 0.5 ml RNA-binding buffer between 240 and 320 nm wavelength range at 20°C with IRF2 5′UTR or mIRF2 5′UTR in absence or presence of purified PTB or bovine serum albumin (BSA).</p

Analysis of translation initiation complex formation in presence of recombinant PTB:

<p>³²P UTP labeled IRF2 UTR was incubated with RRL and amino acid mix and analyzed by sucrose density gradients. Panel A represents the control. Panel B shows the profile of the respective reactions supplemented with either 1.75 or 3.5 nM of purified recombinant PTB protein (as indicated). The panels C–D shows the effect of addition of either GMPPNP (panel C) or Cycloheximide (panel D) used to confirm the 48S and 80S ribosomal peaks (indicated by arrows).</p

RNase VI digestion and Toe-printing of PTB on IRF2 UTR RNA:

<p>(A) IRF2 5′ UTR RNA was incubated with (lane 2) or without (lane 1) of 0.01 units of RNase V1. The unmodified and modified RNAs were reverse transcribed and cDNAs were resolved parallel with a sequencing reaction performed with the same end labeled primer. Cleaved nucleotides are indicated on the right of the panel. (B) For toe-printing, IRF2 wt 5′ UTR RNA was incubated in absence or presence of purified recombinant PTB protein. The RNAs in the Ribonucleoprotein complexes were reverse transcribed and the cDNAs were resolved in 8M urea 8% PAGE in parallel with a sequencing ladder corresponding to IRF2 5′UTR RNA obtained by using the same end labeled primer. The cDNA products terminated at the sites due to protein binding is indicated on the right.</p

Schematic diagram MFOLD structure of IRF2 5′UTR:

<p>Predicted secondary structure of IRF2 5′UTR generated by MFOLD indicating putative contact points of PTB, DMS modification, RNase V1 or T1 cleavage sites.</p