期刊
RNA
卷 17, 期 6, 页码 1120-1131出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.2549411
关键词
tethered hydroxyl radical probing; poliovirus; encephalomyocarditis virus; IRES; RRM (RNA recognition motif); RBD (RNA-binding domain)
资金
- BBSRC [BB/E004857/1, BB/H004203/1]
- Biotechnology and Biological Sciences Research Council [BB/E02209X/1, BB/E004857/1, BB/H004203/1] Funding Source: researchfish
- BBSRC [BB/H004203/1, BB/E004857/1, BB/E02209X/1] Funding Source: UKRI
Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.
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