Using The Yeast Three-hybrid System Screening Human Embryonic Hippocampal Cdna Library In Order To Be Able To Interact With Fmrp Mrnas

The fragile X syndrome is an X linked mental retardation syndrome, characterised by behavioural and physical features such as a long face with large, protruding ears, macro-orchidism, and eye gaze avoidance. The prevalence of the fragile X syndrome was estimated as about 1/4000 for males and 1/6000 for females. Fragile X syndrome results from a mutation (a change in the typical DNA sequence) known as a trinucleotide repeat expansion. This means that a series of three particular nucleotides (CGG) in the DNA is greatly expanded beyond its normal size, disrupting the normal messages that need to be sent. This fact was discovered in 1991 by several teams of researchers studying the X chromosome. In the FMR-1 gene located on the X chromosome, most individuals have a CGG trinucleotide repeat that occurs between 5 and 50 times, the average being around 30. These individuals are normal with respect to fragile X syndrome, and usually carry no risk of transmitting it, although the 40-60 repeat range is sometimes considered a "gray zone" which may or may not be unstable (have a risk of expanding). Some individuals have CGG sequences that are repeated in the range of about 50 to about 200. These individuals are generally referred to as premutation carriers. This means that they carry the syndrome and can transmit it to their children. Premutation carriers, however, are not usually affected by fragile X syndrome. When the number of CGG repeats expands beyond 200, the individual usually has the full mutation. This means that they have fragile X syndrome and will experience the impairments and delays associated with the syndrome.FMRP is an RNA binding protein associated with polyribosomes. A clue for the function of FMRP was given by the finding that FMRP contains motifs (two KH domains and one RGG box) characterizing other RNA binding proteins. Indeed, FMRP is able to bind RNA in vitro, with preference for poly (U) and poly (G) RNA homopolymers. FMRP was shown to colocalize with actively translating polysomes . The association of FMRP to polysomes suggested that it may be involved in the control of mRNA translation. Although some of the messenger RNA targets of this protein, including FMR 1, have been ascertained, many have yet to be identified .Screening methods used to identify the mRNA-targets of FMRP were almost in vitro methods, it's far way from the real situation in living cells. Yeast three-hybrid system is a research method derived from that of yeast two-hybrid system. It provides a technic platform with a stable in vivo environment for the research of these interactions in eukaryotic cells. Although it had been used to study interactions between some potential RNA-targets with FMRP, there is no report about screening work in library scale. In our study, we are planning to screen the RNA-targets of FMRP in library scale using yeast three hybrid system. Finding some new RNA targets is expected.A cDNA library of fetal hippocampus at the age of 19 weeks with the plasmid named pRH5' has been formed for yeast three-hybrid system. The purpose of the present work is to enlarge the size of cDNA library, and use the yeast three-hybrid system to screen the target mRNAs of FMRP in fetal hippocampus. Whole length cDNA of FMR1 were recombined into pYES plasmids, and then transferred into L40-ura yeast strains. The fusion-protein expressed by the pYES plasmids were used as the "bait" to screen the library to find the target mRNAs interacting with FMRP in the yeast cells.About 1.5×10~6 clones have been screened on 120 plates by the X-gal filter paper screen method, and have extracted plasmid from 122 clones revealing positive result in theβ-galactosidase screen. Then the positive clone plasmids were transmitted into TOP10 E.coli., and re-extracted from them. 2,934 plasmids were obtained and sequenced analyzed with two primers respectively, and the sequences analyzed with blasten (NCBI), 50 kinds of pRH5'/cDNA-insert plasmids were identified. Re-transforming these potentially positive plasmids on by one back into the yeast cells followed byβ-galactosidase screening, 31 of 50 were proved to be positive. Up to now, 13 kinds of target-RNAs have been validated by in vitro binding with FMRP and EMSA, 8 of them were proved to be positive. Some of these positive target-mRNAs are associated with the development and differentiation of central nerve system, key-enzymes of some metabolic pathway and some structural proteins et al. Through the above mentioned process, it has been identified that the yeast three-hybrid system is a simple and efficient method in validating the interaction between RNA and protein. As a RNA binding protein, FMRP can interact with many mRNAs acting as a key role in translation, transcription and cell differentiation process. These interactions are closely correlated with the molecular foundation of fragile X syndrome.