The Effects Of Unspliced Intron On Protein Function

Intron splicing is an essential step of eucaryotic mRNA maturation, and is also an important stage of gene expression regulation in eucaryotes. It plays a comprehensive and deep role in regulating gene expresisona and couples tightly with gene transcriptional initiation and elongation, mRNA export, mRNA survailance. It has been reported that intron can influence mRNA translation efficiency and even affects protein stability. In our lab’s previous work, we have produced a series of different Arabidopsis thaliana ribosome protein L36B gene constructs which bear only the first intron with different intron structure and we have transfected them into entogenous RPL36 gene deleted yeast strain FY251 to exam the protein structural and functional differences among Arab.ribosome protein RPL36B produced by these different constructs. Our previous results showed that yeast strains with constructs whose intron can be spliced effectively exhibited a normal growth phenotype and had no defects in 60S large ribosome subunit biogenesis while strains with those constructs whose intron can not be spliced showed a defect in both growth phenotype and 60S large ribosome subunit biogenesis. Here we show that ribosome of the strains with defective 60S large ribosome subunit biogenesis misfunctions in translation initiation and ATG selection and is prone to causing frameshifting during translation. Next we monitor the protein expression level of these different constructs and find that they produce nearly equal amount of Arab.ribosome proteins RPL36B, so it is not because of the lack of Arab.RPL36B protein level that cause the defect in 60S large ribosome subunit biogenesis in strains with unspliced mRNA products. Meanwhlie our Northern Blot result shows that the mRNAs transcripted by unsplicable constructs are unstable and are subjected to Nonsense Mediated mRNA Decay even though they can be exported from nucleus and be translated. We then doubt whether the protein translated by these unstable mRNAs can function normally? To address this question, we performed a rescue experiment:transfect a construct whose intron can be spliced effectively into the strain bearing an unsplicable construct. By doing so, we discover that the defect in growth phenotype of the transfected strain can be recovered. Besides, we find that the original unsplicable construct expresses less Arab. RPL36B protein compared with the corresponding strain which is not rescued while the newly transfected construct expresses the majority of the total protein in the strain and the protein expressed by the newly transfected construct complements the function of the protein expressed by the original unsplicable construct. So we can say that the protein expressed by the unsplicable construct is functionally abnormal. Then we test whether the abnormal protein possesses different translational modifications compared with the functionally normal protein produced by intron splicable construct. By mass spectrometry and Western Blot, our preliminary results show that there is no difference between these two sorts of protein at least on protein acetylation. After that, we hypothesize that the defect in 60S large ribosome subunit biogenesis in strains with unspliced mRNA products are caused by the uneffective assembly of the abnormal protein into ribosome. So we attempted to exam the differences in subcellular distribution between normal protein and abnormal protein and the differences of their behavior during assembly into ribsome and in our experiment we can detect free Arab.RPL36B protein in both strain’s cytoplasm, but both signals are too weak for us to distinguish the difference in quantity. Besides this, we performed SDS-PAGE eletrophoresis and silver staining to detect the protein factors copurified with Arab.RPL36B protein and we hope to find any protein factors that binds differentially to normal Arab.RPL36B protein and/or abnormal Arab.RPL36B protein to provide us with some new clues to explain the functional differences between normal and abnormal protein.With the experimental conditions optimized and the progression of our experiments, we hope to find out the mechanism of how intron splicing influences the protein function which is translated by the unspliced mRNA.