Method to identify IRES elements

Details Method to identify IRES elements Method to identify IRES elements

2002-03-01

2004-12-21

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S006120, C435S039000, C435S334000, C435S320100

Reexamination Certificate

active

06833254

ABSTRACT:

TECHNICAL FIELD
The invention relates to methods to determine whether a particular nucleotide sequence represents an element which behaves as an internal ribosome entry site (IRES). More specifically, the invention employs a bicistronic system involving both cap-mediated and IRES mediated translation.
BACKGROUND ART
Translation of cellular mRNA is generally mediated by a “cap” at the 5′ end of mRNA upstream of the coding sequence in the untranslated region which is responsible for interaction of the messenger RNA with the ribosome. However, certain viruses, including picornaviruses, rhinovirus and HCV have been shown to possess an upstream region, designated IRES, which mediates translation in the absence of the cap structure at the 5′ end of the messenger RNA. This IRES region is typically at least 450 nucleotides long when it occurs in viruses and possesses, at its 3′ end, a conserved UUUC motif followed by a polypyrimidine tract, a G-poor spacer and an AUG triplet. Agol, V., et al.,
Cell
(1992) 68:119-131. The IRES is located within the 5′ untranslated (5′ UTR) region, but downstream from the cap. Certain cellular mRNA's, such as those encoding BiP, c-myc, and eIF4G also contain IRES elements. Johannes, G., et al.,
RNA
(1998) 4:1500-1513, Tsukiyama-Kohara, K., et al.,
J. Virol
. (1992) 66:1476-1483; and Wang, C., et al.,
J. Virol. (
1993) 67:3338-3344.
While viral IRES elements do not require the cap binding protein for complex formation, various other portions of the protein are required for some viral-derived sequences. Pestova, T. V., et al.,
Genes Dev
. (1998) 12:67-83; and Pestova, T. V., et al.,
Mol. Cell. Biol.
(1996) 16:6870-6878. Other trans-acting factors, such as La, PTB, PCBP2, and unr have been shown to interact with viral IRES elements.
The mechanism whereby these IRES elements assist in mediating translation is not known. Little, if any, sequence homology has been found between the various IRES elements (Jackson, R. J., et al.,
RNA
(1995) 1:985-1000). RNA secondary structure appears to play an important role in viral IRES-mediated translation. For example, phenotypic revertants of point mutations in picomaviral IRES elements often include second-site suppressor mutations that restore the wild-type base pairing, suggesting that maintenance of RNA structure is crucial for IRES activity. Also, maintenance of a phylogenetically conserved stem-loop structure was found to be important for the ability of the HCV IRES to mediate translation (Honda, M., et al.,
J. Virol
. (1999) 73:1165-1174). Thus, both sequence and structure are important in viral IRES-mediated translation.
Little is known regarding the cis-acting requirements of cellular IRES elements. It has been proposed that a number of cellular IRES elements possess a Y-type stem-loop-structure, but the ability of these RNA's to mediate translation has not been correlated with maintenance of the Y-shaped structure. Many of the studies investigating cis-acting requirements for IRES elements have focused on mutational analysis of viral 5′UTR's. However, the 5′ regions of viral RNA's are involved in other functions crucial for the viral life cycle, including replication and packaging and cis-acting signals for different viral functions overlap in the 5′ UTR, making it difficult to identify a viral sequence that is solely responsible for IRES-mediated translation.
Recently, the 196 nucleotide 5′ UTR of the mouse Gtx mRNA was found to contain a 9 nucleotide segment that can function as an IRES element. Because this stretch of 9 nucleotides is complementary to 18S rRNA, it was suggested that IRES activity was due to the ability of the 9 nucleotide segment to base-pair with rRNA, thereby recruiting the ribosome to the RNA (Chappell, S. A., et al.,
Proc. Natl. Acad. Sci. USA
(2000) 97:1536-1541). However, comparisons of other IRES elements with rRNA sequences have not, as yet, revealed regions of obvious complementarity.
Robertson, M. E. M., et al.,
RNA
(1999) report an IRES selection system using a cell surface-expressed epitope whose expression is selected for by antibody-coated magnetic beads. A four-base region of the EMCV IRES, proposed to form a GNRA tetraloop, was randomized to a 256 member library and transfected into COS-7 cells. The sequence RNRA was identified as being optimal for translation activity. After three rounds of selection, a maximum of 10-20% of selected clones contained strong IRES elements. At a minimum, the initial library contained 16 positive elements (representing all combinations of the sequence RNRA) out of 256 total elements, thus, at most, a 3-fold increase (from 6% to 20%) in desirable IRES elements was achieved after three rounds of screening.
DISCLOSURE OF THE INVENTION
It has now been shown that although, for example, the native IRES elements contain hundreds of nucleotides, IRES activity can be demonstrated by relatively small segments of RNA, 50 nucleotides in length or less. Segments of RNA containing sequences of this type which exhibit IRES activity or DNA segments which can generate by transcription such RNA segments are useful in the construction of expression systems for proteins in recombinant production. Efficiency of production and assurance of translation can be enhanced by inclusion of these elements in expression systems. Such elements are also useful in screening systems for identifying trans-acting factors that assist in mediating translation.
The invention is directed to materials and methods which permit the identification of nucleotide sequences which represent internal ribosome entry site (IRES) elements. These elements are useful not only in construction of expression systems in a variety of host cells, but are also useful reagents to identify factors which are relevant to translation of RNA's in cells in general.
In one aspect, the invention method employs a bicistronic expression system of at least two modules, a first module which contains a first coding sequence whose translation is mediated by a 5′ cap as a control and a second module which comprises a second coding sequence which has placed, upstream therefrom, a candidate IRES sequence. Typically, the candidate IRES sequence is 100 base pairs or less, more typically 75 base pairs or less, and preferably 50 base pairs or less. Thus, the systems useful in the invention will comprise a DNA which contains a promoter upstream of such a bicistronic system. Typically, the portion of the bicistronic system wherein the translation is cap mediated is placed downstream of the promoter but upstream of the portion of the bicistronic system wherein the translation is mediated by the candidate IRES. However, in an alternative configuration, the cap mediated translation module may be placed downstream of the test module.
Accordingly, in another aspect, the invention is directed to a bicistronic expression system as described above. In another aspect, the invention is directed to a method to identify a nucleotide sequence as an IRES element which method comprises culturing cells or a cell-free system with the expression system described above under conditions where the cap mediated translation occurs and assessing the expression of the module mediated by the putative IRES. Cells or cell-free systems which exhibit expression of the experimental module containing the candidate sequence are thus identified as containing an IRES element. The IRES element can then be recovered and sequenced and used in the manner described above.
In other aspects, the invention is directed to methods to use the IRES elements identified. These applications include methods to treat viral infections in cells, methods to identify trans-acting translation factors, and methods to alter cellular metabolism when said metabolism is controlled by proteins whose expression is mediated by an endogenous IRES element. The invention also includes compositions and kits containing them for use in these methods.


REFERENCES:
patent: 6171821 (2001-01-01), Korne

Affiliated with

Also associated with

Rating

Method to identify IRES elements does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method to identify IRES elements, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method to identify IRES elements will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3323599