The present invention relates to a method of screening potential translational regulatory elements of mRNAs. More specifically, it relates to the method comprising the steps of synthesizing mRNAs which comprise candidates of translational regulatory elements introduced into the untranslated regions (UTRs), and selecting mRNAs with altered translation efficiency by virtue of the inserted motifs.
The present invention also relates to translational regulatory elements screened by the above-described method.
The present invention further relates to a method of isolating a mRNA with altered translation efficiency in comparison with native mRNA by using the above-described method.
It is known that an element, which is present in the 5xe2x80x2-UTR or 3xe2x80x2-UTR of mRNA to control a translation efficiency of the mRNA, plays an important role in regulation of gene expression (Gallie, D. R. and Kobayashi, M., 1994, Gene, 142: 159-165; Yamamoto, Y. et al., 1995, J. Biol. Chem., 270: 12466-12470; Bailey-Serres, J., 1999, Trends Plant Sci., 4: 142-148). The UTR motif is thought to affect a translational initiation frequency by various molecular mechanisms. However, so far there have been known only few examples of analysis for a relation of UTR and translational initiation frequency, and this relation still remains unknown for most genes. In the UTRs there may probably exist various unknown translational regulatory motifs as well as control mechanisms mediated by these motifs. Moreover, there is no example that common properties, motifs, or structures were found among known translational regulatory elements. If there are methods for systematically screening translational regulatory motifs which function in the UTRs, it would enable to clarify basic characteristics of the function essential for the regulatory motifs, or to establish technology to precisely control the expression of a gene of interest at its translation level.
Under the circumstances, the object of this invention is to provide a method of screening potential translational regulatory elements that promote or suppress the translation efficiency of given mRNAs in a translation system, by applying the principles of in vitro evolution.
The inventors have now found a procedure to isolate and analyze a group of UTR regulatory sequences functioning in a given translation system by using an in vitro evolution system of a model gene instead of analyzing UTRs of individual genes one by one. That is, the procedure involves the steps of introducing random nucleotide sequences into UTRs which are potential regulatory sites for gene expression, and repeating the screening of mRNAs that become preferentially translated, or untranslated, in a given translation system due to an effect of the inserted sequence.
Accordingly, the present invention is summarized as follows.
In one aspect of the present invention, the invention provides a method of screening a potential translational regulatory element of mRNA, comprising the steps of synthesizing mRNAs containing random oligonucleotide sequences, which are candidates of translational regulatory elements, introduced into the untranslated regions (UTRs), and selecting mRNA with altered translation efficiency by virtue of the inserted motifs.
In one embodiment of the invention, the selection of the mRNA comprises: (a) introducing a mRNA population, which contains various translational regulatory motif candidates in the UTRs, into an in vitro or in vivo translation system to produce polysomes; and (b) separating a polysomal mRNA with altered translation efficiency.
In another embodiment of the invention, the selection of the mRNA further comprises: (c) extracting RNA from the separated polysomes; and (d) synthesizing a DNA fragment with the RNA as a template, then synthesizing a mRNA with the DNA fragment as a template.
In further embodiment of the invention, the selection of the mRNA further comprises: (e) repeating at least once said steps (a) to (d) for the synthesized mRNA to isolate substantially pure mRNA with altered translation efficiency; and (f) determining a sequence of a potential translational regulatory element introduced into the isolated mRNA.
In further embodiment of the invention, the mRNA synthesized contains either of a Cap structure or a Poly(A) strand, or both. That is, the mRNA is: (1) mRNA containing a Cap structure and a Poly (A) strand, (2) mRNA containing a Cap structure but containing no Poly (A) strand, or (3) mRNA containing no Cap structure but containing a Poly (A) strand. The term xe2x80x9cPoly (A)xe2x80x9d as used herein means polyadenylic acid. The Cap structure is present at the 5xe2x80x2-end of mRNA of a eukaryotic cell or viruse, and it is involved in the initiation reaction for protein synthesis and the like.
In further embodiment of the invention, the in vitro translation system is a cell-free protein synthesis system, and the in vivo translation system is an eukaryotic cell.
In further embodiment of the invention, the separation of polysomes in the step (b) is performed based on the size of polysomes.
In further embodiment of the invention, the mRNA with altered translation efficiency has a higher or lower translation efficiency than that of native mRNA, preferably higher translation efficiency.
Examples of the method of screening according to this invention are as follows:
(1) a method of screening a potential translational regulatory element of mRNA, comprising the steps of:
(a) synthesizing mRNAs containing random oligonucleotide sequences, which are candidates of translational regulatory elements, introduced into the untranslated regions (or UTRs);
(b) introducing the mRNA population synthesized in step (a), which contains various translational regulatory motif candidates in the UTRs, into an in vitro or in vivo translation system to produce polysomes;
(c) separating, based on size, a polysome containing mRNA which has an altered translation efficiency;
(d) extracting RNA from the polysome separated in step (c);
(e) synthesizing a DNA fragment using the RNA extracted in step (d) as a template, then mRNA using the DNA fragment as a template;
(f) repeating at least once the steps (b) to (e) for the mRNA obtained in step (e), and then isolating substantially pure mRNA with altered translation efficiency; and
(g) determining a sequence of a potential translational regulatory element introduced in the mRNA isolated in step (f).
(2) A method of screening a potential translational regulatory element of mRNA, which comprises the steps of:
(a) synthesizing mRNAs containing random oligonucleotide sequences, which are candidates of translational regulatory elements, introduced in the 5xe2x80x2-untranslated region (5xe2x80x2-UTR), and containing a Cap structure and a Poly (A) strand sequence;
(b) introducing the mRNA population synthesized in step (a), which contains various translational regulatory motif candidates in the 5xe2x80x2-UTR, into an in vitro or in vivo translation system to produce polysomes;
(c) separating, based on size, a polysome containing mRNA which has a translation efficiency higher than that of the native mRNA;
(d) extracting RNA from the polysome separated in step (c);
(e) synthesizing a DNA fragment using the RNA extracted in step (d) as a template, then mRNA using the DNA fragment as a template;
(f) repeating at least once the steps (b) to (e) for the mRNA obtained in step (e), and then isolating substantially pure mRNA with translation efficiency higher or lower than that of the native mRNA; and
(g) determining a sequence of a potential translational regulatory element introduced in the mRNA isolated in step (f).
(3) A method of screening a potential translational regulatory element of an mRNA, which comprises the steps of:
(a) synthesizing mRNAs containing random oligonucleotide sequences, which are candidates of translational regulatory elements, introduced in the 3xe2x80x2-untranslated region (3xe2x80x2-UTR), and containing no Cap structure but a Poly (A) stand sequence;
(b) introducing the mRNA population, synthesized in step (a), which contains various translation-regulating sequence candidates in the 3xe2x80x2-UTR, into an in vitro or in vivo translation system to produce polysomes;
(c) separating, based on size, a polysome containing the mRNA which has a translation efficiency higher than the native mRNA;
(d) extracting RNA from the polysome separated in step (c);
(e) synthesizing a DNA fragment using the RNA extracted in step (d) as a template, then mRNA using the DNA fragment as a template;
(f) repeating at least once the steps (b) to (e) for the mRNA obtained in step (e), and then isolating substantially pure mRNA with translation efficiency higher or lower than that of the native mRNA; and
(g) determining a sequence of a potential translational regulatory element introduced into the mRNA isolated in step (f).
In another aspect of the present invention, the invention provides a method of screening a translational regulatory element of a native mRNA with high translation efficiency, comprising the steps of:
(a) introducing a mRNA population, which contains various translational regulatory motifs in either or both of the untranslated regions, into an in vitro or in vivo translation system to produce polysomes;
(b) separating based on the size a polysome containing the mRNA which has a high translation efficiency;
(c) extracting an mRNA from the polysome separated in step (b);
(d) synthesizing a DNA fragment using the RNA extracted in step (c) as a template, then mRNA using the DNA fragment as a template;
(e) repeating at least once the steps (a) to (d) for the mRNA obtained in step (d), and then isolating substantially pure mRNA with high translation efficiency; and
(f) determining a sequence of a translational regulatory element of the mRNA isolated in step (e).
The present invention, in further aspect thereof, provides a translational regulatory element comprising a sequence selected from the group consisting of sequences represented by SEQ ID NOS: 9 to 28.
In yet another aspect, the present invention provides a method of isolating an mRNA with altered translation efficiency in comparison with natural (or native or intact) mRNA by using any one of the methods as described above. Specifically, the method comprises the steps of:
(a) synthesizing mRNAs containing random oligonucleotide sequences, which are candidates of translational regulatory elements, introduced into the untranslated regions (or UTRs);
(b) introducing the mRNA population synthesized in step (a), which contains various translational regulatory motif candidates in the UTRs, into an in vitro or in vivo translation system to produce polysomes;
(c) separating, based on size, a polysome containing mRNA which has an altered translation efficiency;
(d) extracting RNA from the polysome separated in step (c);
(e) synthesizing a DNA fragment using the RNA extracted in step (d) as a template, then mRNA using the DNA fragment as a template; and
(f) repeating at least once the steps (b) to (e) for the mRNA synthesized in step (e), and then isolating substantially pure mRNA with altered translation efficiency.
The mRNA of the step (a) above can contain either a Cap structure or a Poly(A) strand, or both.