Polypyrimidine tract‐binding protein binds to the 5′ untranslated region of the mouse mammary tumor virus mRNA and stimulates cap‐independent translation initiation

Abstract

The 5 0 untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5 0 UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.

SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies

“…In this study, SINEUP-GFP showed direct interaction with 40S, implying that a multimer made up of SINEUP RNA, PTBP1, 40S and the target mRNA may contribute to target mRNA stabilization. There are several reports that PTBP1 is recruited during cap-independent translation to stimulate translational initiation, by re-modelling the target transcripts followed by supplying the small subunit (40S) to binding sites ( 37 , 38 ). In one example of translation initiation, the cricket paralysis virus (CrPV) recruits the 40S ribosome subunit to its RNA with high affinity, and requires eukaryotic elongation factor (eEF)1A and eEF2 to recruit tRNA for assembly of 80S tertiary complexes ( 39 ).…”

SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies

“…LARP1, which is known as 5'TOP mRNA binding proteins, stabilize the target 5'TOP mRNAs by forming complex with 40S ribosome subunit (Gentilella et al, 2017), In this study, SINEUP-GFP showed direct interaction with 40S, implying that SINEUP RNA with multimerization including PTBP1, 40S and the target mRNA may contribute the target mRNA stabilization. There are several reports that PTBP is recruited to cap-independent translation to stimulate translational initiation with re-modelling the target transcripts followed by supplying the small subunit (40S) to binding sites (Caceres et al, 2016;Stoneley & Willis, 2004). In one example of translational initiation mechanism, the cricket paralysis virus (CrPV) recruits 40S ribosome subunit on its RNA with high affinity, and requires eukaryotic elongation factor (eEF)1A and eEF2 to recruit tRNA for assembly of 80S tertiary complexes (Johnson et al, 2017).…”

Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

“…UV-crosslinking immunoprecipitation (CLIP) was performed as described in ( 35 ), using HEK 293T cells transfected with the dl HIV-1 IRES plasmid (7.5 μg). Four hours post-transfection, cells were treated with, or without, 10 μM of EPZ015666 and 48 h later cells were washed, covered with of cold PBS 1× (7 ml/10 cm plate), and UV-irradiated (UV-254 nm, 400 mJ/cm 2 ) on ice in a UV chamber (UVP-CL-1000, Upland, CA, USA).…”

“…Four hours post-transfection, cells were treated with, or without, 10 μM of EPZ015666 and 48 h later cells were washed, covered with of cold PBS 1× (7 ml/10 cm plate), and UV-irradiated (UV-254 nm, 400 mJ/cm 2 ) on ice in a UV chamber (UVP-CL-1000, Upland, CA, USA). Cells were scraped, collected by centrifugation at 4°C, and lysed using RIPA buffer (Tris–HCl pH 7.5 10 mM, EDTA 1 mM, NaCl 150 mM, NP-40 0.5%, sodium deoxycholate 0.5%, SDS 0.1%, plus Roche protease inhibitors), 3 μl Riboblock (Fermentas) and 6 μl DNAseRQ1 (Promega) and treated as detailed in ( 35 ). The cell lysate was precleared with BSA pre-blocked protein-A/G-Agarose beads (#SC-2003, Santa Cruz Biotechnology Inc, Dallas, TX, USA) ( 35 ).…”