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

Association of Dengue Virus Serotypes 1&2 with Severe Dengue Having Deletions in Their 3′Untranslated Regions (3′UTRs)

Dengue virus infections are recorded as hyper-endemic in many countries, including India. Research pertaining to the reasons for frequent outbreaks and severe dengue is ongoing. Hyderabad city, India, has been recorded as a ‘hotspot’ for dengue virus infections. Dengue virus strains circulating over the past few years in Hyderabad city have been characterized at the molecular level to analyze the serotype/genotypes; 3′UTRs were further amplified and sequenced. The disease severity in patients infected with dengue virus strains with complete and 3′UTR deletion mutants was analyzed. Genotype I of the serotype 1 replaced genotype III, which has been circulating over the past few years in this region. Coincidentally, the number of dengue virus infections significantly increased in this region during the study period. Nucleotide sequence analysis suggested twenty-two and eight nucleotide deletions in the 3′UTR of DENV-1. The eight nucleotide deletions observed in the case of DENV-1 3′UTR were the first reported in this instance. A 50 nucleotide deletion was identified in the case of the serotype DENV-2. Importantly, these deletion mutants were found to cause severe dengue, even though they were found to be replication incompetent. This study emphasized the role of dengue virus 3′UTRs on severe dengue and emerging outbreaks

Differential localization of dengue virus protease affects cell homeostasis and triggers to thrombocytopenia

Summary: Thrombocytopenia is one of the symptoms of many virus infections which is the “hallmark” in the case of dengue virus. In this study, we show the differential localization of existing two forms of dengue virus protease, i.e., NS2BNS3 to the nucleus and NS3 to the nucleus and mitochondria. We also report a nuclear transcription factor, erythroid differentiation regulatory factor 1 (EDRF1), as the substrate for this protease. EDRF1 regulates the expression and activity of GATA1, which in turn controls spectrin synthesis. Both GATA1 and spectrins are required for platelet formation. On the other hand, we found that the mitochondrial activities will be damaged by NS3 localization which cleaves GrpEL1, a co-chaperone of mitochondrial Hsp70. Levels of both EDRF1 and GrpEL1 were found to deteriorate in dengue virus-infected clinical samples. Hence, we conclude that NS2BNS3-mediated EDRF1 cleavage and the NS3-led mitochondrial dysfunction account for thrombocytopenia

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

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

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

Deletion of 3′ end IRF2 UTR RNA showed reduced activity:

<p>(A) Schematic representation of bicistronic construct used for translation studies. (B) Bicistronic plasmid (1 µg) either pRΔEIRF2F or pRΔE3′δ25IRF2F were transiently transfected into HeLa cells. 16 hrs post transfection, respective luciferase activities corresponding to Fluc and RLuc were measured and shown separately. Transfection efficiencies were normalized by co-transfecting with a β-galactosidase plasmid. The data represents mean±s.d. from three independent experiments.</p

UV Crosslinking of PTB with wt IRF2 5′UTR and 3′ end deletion mutant:

<p>(A) A schematic representation of wt and mIRF2 UTRs used in UV-cross linking and translation studies. (B) Purified PTB was incubated with ³²PIRF2 5′UTR (lanes 2–4) or with ³²P mIRF2 5′UTR RNA (lanes 5–7). The RNA protein complexes were UV cross linked and analyzed on SDS 10% PAGE followed by phosphorimaging. Lane N represents no protein control. (C) Purified recombinant PTB protein was incubated with ³²P IRF2 5′UTR in absence (lane 2) or presence of 100 and 200 fold molar excess of self cold IRF2 5′UTR (Lanes 3–4) or mIRF2 RNA (lanes 5–6). The RNA protein complexes were UV cross linked and analyzed on SDS 10% PAGE followed by phosphorimaging. Lane N represents no protein control. The bands were quantified using densitometric analysis and the values have been represented below the panel.</p

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