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Characterization of immunophilins in the silkmoth Bombyx mori
J A Somarelli1, J L Coll, A Velandia
1Department of Biological Sciences, OE304, Florida International University, Miami, Florida 33199, USA.
Abstract:
The FK506-binding proteins (FKBPs) perform an extensive variety of functions in numerous organisms from archaea to humans. The FKBPs are distinguished by their peptidyl-prolyl cis-trans isomerase (PPIase) activity and ability to bind the immunosuppressive drugs FK506 and rapamycin. Here, we report the isolation and characterization of FKBP45, a novel member of the FKBP family obtained from U1 small nuclear RNA (snRNA) binding assays using Bombyx mori nuclear extracts. The protein, an apparent orthologue of FKBP46 from the armyworm, Spodoptera frugiperda, was found to associate with U1 stem-loop I RNA in vitro. The FKBP45 cDNA was isolated and the genomic sequence was characterized, including the positions of exon/intron junctions and consensus splice sites. Using bioinformatics, transcription factor consensus binding sites were identified and subsequent Western blotting from developing eggs indicate that FKBP45 is differentially expressed during embryogenesis. A database was assembled using more than 1,800 available FKBP amino acid sequences and pairwise sequence alignments revealed several putative FKBP45 orthologues in various species. Analysis of these sequences revealed the position of an RNA binding domain within this new protein. In addition, FKBP45 possesses similar characteristics to several potential orthologues, including the presence of bipartite nuclear localization signals (NLSs) and phosphorylation sites.
Insights
Researchers identified FKBP45, a novel FK506-binding protein (FKBP) that binds U1 small nuclear RNA (snRNA). This protein is differentially expressed during embryogenesis and contains an RNA-binding domain, suggesting a role in RNA processing.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- FK506-binding proteins (FKBPs) are crucial enzymes with peptidyl-prolyl cis-trans isomerase (PPIase) activity.
- FKBPs are involved in diverse cellular functions across various organisms.
- Their ability to bind immunosuppressive drugs like FK506 and rapamycin is a key characteristic.
Purpose of the Study:
- To isolate and characterize a novel FKBP member, designated FKBP45, from Bombyx mori.
- To investigate the association of FKBP45 with U1 small nuclear RNA (snRNA).
- To analyze the genomic sequence, expression patterns, and evolutionary relationships of FKBP45.
Main Methods:
- U1 small nuclear RNA (snRNA) binding assays using Bombyx mori nuclear extracts.
- cDNA isolation and genomic sequence characterization.
- Bioinformatic analysis of transcription factor binding sites and sequence alignments.
- Western blotting to assess protein expression during embryogenesis.
Main Results:
- FKBP45 was isolated and characterized as a novel FKBP family member.
- FKBP45 was found to associate with U1 stem-loop I RNA in vitro.
- The genomic sequence, including exon/intron boundaries, was determined.
- Differential expression of FKBP45 during embryogenesis was observed.
- Bioinformatic analysis revealed an RNA binding domain, bipartite nuclear localization signals (NLSs), and phosphorylation sites in FKBP45.
- Putative FKBP45 orthologues were identified across various species.
Conclusions:
- FKBP45 is a novel FKBP with demonstrated RNA-binding capabilities.
- Its differential expression during embryogenesis suggests a significant role in developmental processes.
- The identified RNA binding domain and NLSs indicate potential involvement in nuclear RNA metabolism.
