Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regul ating protein levels. One such element is the internal ribosome entry site (IRES), We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES, To identify other short sequences with similar properties, we designed a selec tion procedure that uses a retroviral vector to express dicistronic mRNAs e ncoding enhanced green and cyan fluorescent proteins as the first and secon d cistrons, respectively. Expression of the second cistron was dependent up on the intercistronic sequences and was indicative of IRES activity, B104 c ells were infected with two retroviral libraries that contained random sequ ences of 9 or 18 nt in the intercistronic region, tells expressing both cis trons were sorted, and sequences recovered from selected cells were reassay ed for IRES activity in a dual luciferase dicistronic mRNA, Two novel IRESe s were identified by this procedure, and both contained segments with compl ementarity to 18S rRNA. When multiple copies of either segment were I in ke d together, IRES activities were dramatically enhanced. Moreover, these syn thetic IRESes were differentially active in various cell types. These prope rties are similar to those of the previously identified 9-nt IRES module fr om Gtx mRNA, These results provide further evidence that short nucleotide s equences can function as IRESes and support the idea that some cellular IRE Ses may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the desig n of vectors for biotechnology and gene therapy.