Analysis On Changes Evolved In The Mechanisms Of Intron Splicing From Yeast To Plant-Using Splicing Of The Arabidopsis Ribosomal Protein RPL36B First Intron In Yeast As An Example (Ⅱ)

Recent studies make us have a deeper understanding of intron’s function and meaning, but it raises new problems.The experimental results show that intron can influence transcription, mRNA modification, mRNA stability, transport, translation efficiency and so on different steps of gene expression. To understand these efferts, we maybe know the mechanism of intron splicing from a broader perspective.The laboratory preliminary results show that most of the plant intron can not be effectively recognized and spliced in yeast cells, we hope to explore corresponding changes of the impact on cis-elements and trans-factors in intron splicing from yeast evolution to plant evolution for understanding elements involved in intron splicing.In order to keep the original structure and physiological function of the target gene,we construct a series of simplified Arabidopsis ribosomal protein RPL36B mutant gene and transform them into cell missing endogenous Yeast RPL36B to make Arabidopsis RPL36B become unique RPL36B protein sources to observe effect of Arabidopsis mutant gene on cell growth and their splicing efficiency.It has been reported that sequence surrounding intron, intron GC content, exon GC content, promoter and snRNA with its associated protein have a different impact on intron splicing efficiency. Based on the above analysis, we made a study as follows:first, in order to analyze the sequence surrounding intron effect on intron splicing efficiency; we transfer a simplified series of Arabidopsis ribosomal protein RPL36B mutant gene from 5’untranscriptional regions (5’UTR) into the coding region without creating new endonuelease sites. We find that only the construct result has transformed from spliced completely into spliced uncompletely whose 5’ss and BS are mutated into Yeast’5’ss and BS simultaneously by RT-PCR. Preliminary results show that sequence arounding the intron has little distinct effect on its splicing efficiency. Next, to determine GC content and exon GC content’s impact on intron splicing efficiency, we replace Arab first intron with High GC yeast intron and Low GC yeast intron respectively, we also have make a series of point mutation on its 5’ss alone and 5’ss together with BS.We find that different GC content of yeast intron can be spliced completely by RT-PCR.However, with respect to the Arabidopsis intron, it is can be incompletely spliced when we mutate 5’ss alone; it can not be spliced while 5’ss and BS of intron was mutated at the same time by RT-PCR. According to these result, we infer that the 5’ss and branch site play a key role in the splicing of intron and the GC content has no significant effect on the splicing of intron. To have a more systematic understanding on intron splicing,we analysis promoters’effect on intron splicing; we have replaced Yeast promoter with ADH1 promoter to illustrate different promoters effect on the intron spicing efficiency. We find the promoter almost cannot affect the intron splicing by RT-PCR, so the most important factor of determing the intron splicing is intron recognition. Finally,we research intron splicing from snRNA affecting intron recognition.It is well known that U1 snRNA indentify the 5’ss splicing sites of intron with complementary base pairs and the U2 snRNA indentify the branch sites of intron in the same way. So the 5’ss and branch sites of intron synergistic identification play a key role in intron spicing. Given the conserved differences in Arabidopsis and yeast intron splice sites, suggesting that in Arabidopsis snRNAs Ul and U2 in the splice site recognition process has a higher plasticity, in particular in the branch point. For the purpose of that, we want to import Arabidopsis U1 snRNA and U2 snRNA into yeast cells to see whether it can promoter Arabidopsis intron splicing in yeast. We haved cloned Arabidopsis U1 snRNA and U2 snRNA and then transferred it into B, B +3M and 5M,5M+3M yeast mutant strains removing endogenous RPL36B. But preliminary testing results show that they have unconspicuous effect with by spotting assay and RT-PCR. We have designed the specific Arabidopsis reverse transcription primers and PCR primer to determine whether U1 snRNA and U2 snRNA have no sufficient transcription or it is not enough facilitate for intron spicing only with the help of U1 snRNA and U2 snRNA. It is determined that the U1 snRNA and U2 snRNA is not enough to promote intron splicing, it also needs some splicing associated protein. We will transform Arabidopsis specific protein P14, SF1 and SF3B155 together with snRNA into yeast mutant strains.At the same time, we also plan to contruct Arabidopsis and yeast cDNA library, and hope to screen some factors to promote the Arabidopsis intron splicing.From the need for additional cis-and trans elements for Arabidopsis intron splicing in yeast cells, we have a better understanding the mechanism and significance of intron splicing from the evolution of yeast to Arabidopsis.