The following information accompanied stocks donated to the Bloomington Stock Center by Paula Vazquez-Pianzola and Beat Suter, University of Bern.
P{PTT-un}G00080 lies in an intron upstream of the most 5’ exon shared by all Imp splice isoforms. It adds an internal EGFP tag to Imp proteins with a variable number of amino acids N-terminal to the tag based on the use of alternative upstream exons.
Mutations were introduced by CRISPR mutagenesis onto the P{PTT-un}G00080 chromosome as follows.
The sequence changes in ImpKH1 result in substitution of GDDG for GRQG in the KH1 RNA-binding domain.
The sequence changes in ImpKH2 result in substitution of GDDG for GKSG in the KH2 RNA-binding domain.
The sequence changes in ImpKH3 result in substitution of GDDG for GTRG in the KH3 RNA-binding domain.
The sequence changes in ImpKH4 result in substitution of GDDG for GKGG in the KH4 RNA-binding domain.
In these four cases, RNA binding by the specific domain was eliminated. All homozygotes were viable and fertile and had no clear mutant phenotypes.
The sequence changes in ImpKH1-2 result in substitution of GDDG for GXXG in the KH1 and KH2 RNA-binding domains (as above for the individual domains).
The sequence changes in ImpKH3-4 result in substitution of GDDG for GXXG in the KH3 and KH4 RNA-binding domains (as above for the individual domains).
In these two cases, homozygotes were lethal and Imp protein was mislocalized.
Imp25-1A6 was also induced on the P{PTT-un}G00080 chromosome by CRISPR mutagenesis. A small deletion after the inserted P{PTT-un} element results in a frameshift to generate N-terminal, EGFP-tagged peptides. For example, the Imp-RA splicing pattern results in a peptide of the form
31 aa of IMP + EGFP + 63 aa of IMP + 18 aa coded by out-of-frame codons after the deletion
or
MHSNNNSSRLNNNISNNYYQQKQSLIRYLDR(GFP)AAVGLNGVEFEGSKLHAEQLDKNQRRSQRNQRNPYPGMPGPGRQADFPLRILVQSEMVGAIIGqhhqddhttepcsrrrps
The nucleotide changes associated with the amino acid substitutions in ImpKH1, ImpKH2, ImpKH3, and ImpKH4 are shown in the associated file. ImpKH1-2 and ImpKH3-4 are just a combination of the single mutants.