Premium
Studying the Effects of MDS Mutations in HSH155 and PRP21 on the Splicing of Introns
Author(s) -
Luo George,
Carrocci Tucker,
Zoerner Douglas,
Xu Jiacui,
Hoskins Aaron
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.593.2
Subject(s) - spliceosome , rna splicing , snrnp , intron , biology , prp24 , genetics , gene , saccharomyces cerevisiae , exon , messenger rna , splicing factor , alternative splicing , mutation , microbiology and biotechnology , rna
The spliceosome is responsible for the excision of introns from pre‐mRNA in eukaryotes and alterations of the spliceosome can cause serious consequences. Recently, studies have shown mutations in certain genes associated with the spliceosome are linked with certain types of Myelodysplastic Syndromes (MDS). Specifically, mutations in SF3B1 , an U2snRNP associated splicing factor, have been found in approximately 20% of MDS cases. SF3B1 is thought to bind pre‐mRNA and to help link U2snRNP to pre‐mRNA. In our research, we investigated the corresponding mutations in the S. cerevisiae homolog, HSH155 , which is well‐conserved between humans and yeast. We have introduced MDS‐related mutations into S. cerevisiae HSH155 and showed the cells have no significant growth differences or temperature sensitive defects compared to the wild type. We expected splicing will be affected by the mutations, resulting in an increased ratio of pre‐mRNA to mRNA. To check this hypothesis, we carried out Reverse‐Transcription Polymerase Chain Reaction (RT‐PCR) but found no differences in splicing efficiency of tested pre‐mRNAs ( SUS1, DYN2, GLC7, YFR045w, PMI40, MATa1 ) in S. cerevisiae . Our results suggest that changes in splicing due to SF3B1 mutations may not be apparent when studying endogenous yeast pre‐mRNAs. Simultaneously we are investigating the effects of mutations in a second spliceosomal protein associated with MDS cases, PRP21p. PRP21p is homologous to the human SF3A1 protein and is also associated with the U2 snRNP. For this project, we have produced the necessary yeast strains and have used growth assays to check for growth differences and temperature sensitivity as well as RT‐PCR experiments to test for splicing of endogenous genes. Additionally, we will use an ACT1‐CUP1 copper reporter gene that will help us determine splicing efficiency in cells. Through this assay, we will determine if splicing of wild type and mutated intron sequences are affected by mutations in PRP21 .