Evidence that Noncoding RNA dutA Is a Multicopy Suppressor of Dictyostelium discoideum STAT Protein Dd-STATa

Abstract

Dd-STATa, a Dictyostelium discoideum homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed. There were 12 multicopy suppressors of Dd-STATa, of which 4 encoded segments of a known noncoding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATa is activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosinephosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknown mechanism.

Dictyostelium Finds New Roles to Model

“…Although valuable results have been obtained using a Dictyostelium cDNA expression library (Robinson and Spudich 2000;Shimada and Kawata 2007;Shimada et al 2008), the library is very unlikely to contain full-length sequences representing all the less highly transcribed genes. The alternative approach of genomic DNA complementation has never been made to work, probably because of the difficulty of amplifying representative genomic Dictyostelium DNA libraries in E. coli.…”

GBF-dependent family genes morphologically suppress the partially active Dictyostelium STATa strain

“…In general, however, which molecules regulate Dd-STATa activity and which genes are regulated by Dd-STATa remain poorly understood. To search for molecules linked to the STAT signaling pathway, we previously identified 13 potential Dd-STATa target genes using in situ hybridization (Shimada et al 2004a, b;Aoshima et al 2006) and isolated suppressor clones by genetic screening (Shimada and Kawata 2007). We report herein that a putative suppressor clone, NK20, suppresses the partially active STATa strain, at least morphologically, when it is overexpressed.…”

“…Creation of D. discoideum strains used for genetic screening having an integrated relicase gene and a green fluorescent protein (GFP)-fused N-terminal truncated DdSTATa core fragment, GFP-STATa(core)::ORF+, construction of the cDNA library from slug stage and screening the Dd-STATa multicopy suppressors are described in our previous paper (Shimada and Kawata 2007).…”

“…Then they were allowed to develop on thin water agar poured onto slide glass plates for 38 h at 22°C. After 38 h, the slugs and fruiting bodies (including culminants) were counted and the fruiting body formation ratio was calculated as described previously (Shimada and Kawata 2007).…”

“…The latter technique also increases the richness of the approach because various genetic interactions can be included: overexpression, gain of function, dominant-negative and attenuated functions (Girard et al 2004;Octtaviani et al 2006;Robinson and Spudich 2000). Using this approach, we recently isolated multicopy suppressor clones of Dd-STATa, a functional homologue of metazoan signal transducers and activators of transcription (STAT) proteins (Shimada and Kawata 2007).…”