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Detailed mapping of the sites of interaction of polypyrimidine tract binding protein with its RNA targets: viral and cellular IRESs and pre-mRNAs.

Reference	BB/E004857/1
Principal Investigator / Supervisor	Professor Richard Jackson
Co-Investigators / Co-Supervisors	Dr Ann Kaminski
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	286,334
Status	Completed
Type	Research Grant
Start date	10/01/2007
End date	09/01/2010
Duration	36 months

Abstract

A cDNA clone suitable for bacterial expression of His-tagged PTB will be mutated to give at least 8 variants, each with a single surface-exposed cysteine residue close to one of the four RBDs (a minimum of two variants with a single cysteine proximal to each RBD). The recombinant proteins will then be conjugated with Fe(II)-BABE (iron-p-bromoacetamidobenzyl EDTA). After verifying that these Fe(II)-BABE conjugates retain in vitro biological activity towards their RNA targets, they will be used for tethered hydroxyl radical footprinting. This will involve incubating the conjugated PTB mutants individually with 32P-labelled target RNA, initiating the Fenton reaction by addition of ascorbic acid/hydrogen peroxide, and then determining the sites at which the target RNA has been cleaved by the resulting hydroxyl radicals. By this approach, we will be able to define which RNA segments are contacted by each of the 4 RBDs, i.e. we will be able to 'dock' the PTB on to the RNA sequence. The next stage will be to test whether all these contacts are necessary for biological activity. To this end, mutations will be introduced into the RNA binding surface of each RBD. The in vitro biological activity of these mutants will then be assessed, and hydroxyl radical footprinting used to verify that the interaction of the mutated RBD with the RNA has been disrupted without perturbing the interactions of the other 3 RBDs. These approaches will be applied to the interaction of PTB with the following targets: 1) the encephalomyocarditis virus IRES (internal ribosome entry site); 2) the IRESs of poliovirus (an enterovirus) and human rhinovirus; 3) the exons (plus flanking introns) of alpha-actinin and alpha-tropomyosin pre-mRNAs that are subject to alternative splicing regulated by PTB; 4) a selection of cellular mRNA IRESs that are known to be stimulated by PTB: Apaf-1 and BAG-1 mRNA IRESs.

Summary

The strong physical, behavioural, and mental similarity of identical twins shows that it is our genetic material that largely specifies what we are. This genetic material is our DNA, which can be regarded as an exceedingly long 'tape' (e.g. an enormous video tape) with some 3 billion bits of information, coding for the numerous different types of protein which carry out most of our bodily functions. The DNA can be thought of as the 'master tape', an archive of thousands of normal length videos joined together. The process of decoding this information in the DNA involves first making a working copy of one of these videotapes in the archive. In fact the information in this initial copy is segmented, with the meaningful segments interspersed with meaningless sections. Consequently, the meaningful segments in this initial copy need to be cut out and spliced together, much as cine films were created by splicing in the pre-electronic era. In many cases, this splicing can occur in slightly different ways, with some meaningful segments excluded in some cells or some circumstances, but included in other cells/circumstances. Thus the initial copy of the archived 'video' can give rise to more than one variant of the final playable video. A protein known as polypyrimidine tract binding protein (PTB) exerts a strong influence on the alternative splicing, causing the inclusion of some meaningful segments and the exclusion of others. After the video has been spliced together it is decoded by a small particle known as a ribosome, which essentially carries out an analogous function to that of a video recorder (VCR) as it decodes the information as pictures. The 'biological videos' are similar to real videos in so far as the part of the tape with the pictorial information is preceded by a short leader length that has no information. In most cases our biological VCR (the ribosome) finds the point where the true information starts by searching or scanning in fast-forward mode through the whole meaningless leader. However, with some such videos the biological VCR is able to go straight to the starting point where the meaningful pictorial information begins without searching through the meaningless leader section, and in many such cases PTB is necessary for the direct location of the start position by the biological VCR. The aim of this project is to find out how PTB (a) causes the biological VCR to locate these starting sites without a need for searching through the tape from the very start, and (b) can influence the exact pattern in which the different meaningful segments are spliced together to give the finished video tape.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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