Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-10-23
2003-10-21
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S006120, C435S007100
Reexamination Certificate
active
06635440
ABSTRACT:
The present invention provides a method of isolation of compounds that bind to ribonucleic acid (RNA). In particular, this method is suitable for use in identifying compounds for which a cognate RNA binding site is known. This method allows the isolation of new compounds that bind specifically to RNA and may thus be used in the study of the interactions that are important for the normal function of the cell or for the activity of viruses. This method may also be used to identify RNA binding compounds with potential efficacy as pharmaceuticals.
Proteins have important roles in the structure and function of all types of RNA (mRNA, rRNA, tRNA and viral RNA), ranging from packaging nucleic acid molecules in a stable structural configuration to mediating all aspects of RNA metabolism. Many interactions between RNA and proteins are specific for consensus recognition sites defined by the nucleic acid sequence of the mRNA. It is these interactions in particular that often have profound effects on the relative stabilities of certain RNA species, or that affect the rate and degree of translation from these mRNAs and their locations within a cell.
A rapidly increasing number of examples provide evidence that gene expression can be regulated in the cytoplasm of eukaryotic cells at the level of mRNA stability, translation, or the subcellular localisation of mRNAs (reviewed by Sachs 1993; Merrick 1992). The biological processes controlled by such mechanisms range from biological processes as diverse as early embryonic development, sexual differentiation, intelligence and viral replication. Common to these post-transcriptional methods of control is the role played by specific interactions between cis-acting sequences in the mature mRNAs and regulatory binding proteins. Most commonly, the regulatory sequences are contained within the untranslated regions at the 5′ and 3′ ends of the mRNA transcripts.
In prokaryotes, numerous examples of post-transcriptional regulation by repressor proteins exist. In most cases, the repressor proteins directly or indirectly occlude the Shine-Dalgarno sequence and/or the initiation codon.
In eukaryotes, one of the best examples of specific interactions between RNA and binding proteins that has been elucidated to date is the regulation of ferritin and erythroid 5-aminolevulinate synthase mRNAs by iron regulatory proteins (IRPs). IRP1 or IRP2 bind to the iron responsive element (IRE) in the 5′ untranslated region of a mRNA in a position proximal to the CAP binding site and controls the initiation step of translation. It is a mutation in an IRE in the ferritin L-chain mRNA that leads to hyperferritinaemia/cataract syndrome in human patients (Beaumont et al, 1995).
More recently, further examples have been identified in eukaryotic cells, such as the L32 yeast ribosomal protein (Dabeva and Warner, 1993) and proteins acting on Drosophila spermatogenesis that are further candidates for repressors that are functionally similar to IRP. Examples have also been found that bind further downstream from the CAP structure, such as thymidylate kinase (Chu et al., 1993). LOX-BP binds to the 3′ region of 15-lipoxygenase mRNA and represses its translation (Ostareck-Lederer et al, 1994).
The biological importance of RNA binding protein-binding site effector pairs means that a useful mechanism is needed by which these RNA binding proteins can be identified and further examined. Such a strategy had not proven easy to devise, partly because, in many cases, RNA-binding proteins exert their functions in cells or tissues that are not readily amenable to biochemical analysis, such as specific areas of the brain or in the germ line. The unavailability of biochemical material imposes cumbersome limitations on the identification and cloning of biologically important RNA-binding proteins operating in such systems, particularly when compounded by a lack of possible genetic approaches.
To circumvent such limitations, alternative strategies to identify and study RNA-protein interactions have been devised, based on phage display (Laird-Offringa et al., 1995), transcription termination (Harada et al., 1996) or translation in
E. coli
, (Jain and Belasco, 1996) or on transcription initiation in yeast (SenGupta et al., 1996; Putz et al, 1996).
One approach to study RNA-protein interactions has previously been developed by the present inventors. This approach is based on the realisation that the binding of protein to specific sites near the 5′ end of an mRNA molecule causes its translation to be repressed both in mammalian cells and in yeast (Stripecke and Hentze, 1992; Stripecke et al., 1994; Gray and Hentze, 1994). RNA binding proteins with physiological functions unrelated to eukaryotic mRNA translation were found to function as translational repressor proteins when their specific cognate binding site was introduced into the 5′UTR in a position similar to that of the IRE in ferritin mRNA (Stripecke et al., 1994).
Due to the step-wise nature of the translational initiation process, in which ribosome subunits and cofactors must have access to its their binding sites on the mRNA molecule, translational repression using the Stripecke method appears not to be restricted by the normal physiological function of the RNA-binding protein used. The Stripecke method thus allows the study of almost any protein-RNA binding site pair.
In the Stripecke method, the cognate binding sites for the bacteriophage protein MS2-CP and the spliceosomal component U1A were each cloned into the 5′UTR of luciferase indicator mRNAs using a restriction enzyme site nine nucleotides downstream of the major transcription initiation site. The repressor proteins were cloned under an inducible GAL4/PGK promoter so that luciferase activity could be assayed in the presence and absence of binding protein. It was found that in the presence of both the cognate binding protein and the RNA binding site in the luciferase mRNA, luciferase activity decreased in correlation with the affinity between the binding protein and the binding site in the mRNA. This correlation was also shown to exist in HeLa cells. The translation of the luciferase indicator constructs could be decreased up to ten-fold by this mechanism of repression.
This system thus allows the study of the affinities between specific RNA binding sites and cognate positions. However, in order to be able to assay for luciferase activity the cells need to be harvested and lysed. This means that no linkage between phenotype (i.e. repression of translation caused by the presence of an efficacious binding protein) and genotype (the gene which coded for the active binding protein) is retained. This method is therefore only suited to the assessment of the degree of affinity with which a specific binding pair interact and essentially does not allow the isolation of novel RNA binding proteins or their coding genes.
There is thus a great need for a method of detection of nucleic acids that code for RNA binding compounds, particularly proteins.
SUMMARY OF THE INVENTION
According to the present invention there is provided a method for the isolation of nucleic acid coding for a RNA-binding compound, said method comprising the steps of:
a) expressing in a host cell
i) one or more candidate nucleic acids to be screened for coding for the RNA binding compound, and
ii) a nucleic acid comprising a coding region for a fluorescent marker protein, wherein the nucleic acid comprising the coding region for the fluorescent marker protein includes a binding site for the RNA-binding compound,
b) selecting a host cell that exhibits an altered level of fluorescence from levels found in a host cell containing ii) alone; and
c) isolating the nucleic acid coding for the RNA binding compound from the selected cell.
This approach is referred to as TRAP (Translational Repression Assay Procedure) and makes use of the discovery that the binding of a compound to a sequence that is present in a mRNA species prevents its translation. The mechanism of this prevention is thought to
Hentze Matthias W.
Paraskeva Efrosyni
Conlin David G.
Edwards & Angell LLP
European Molecular Biology Laboratory
Loeb Bronwen M.
Rees Dianne M.
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