ABSTRACT
NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.
Subject(s)
Methyltransferases/metabolism , Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Cell Nucleolus/drug effects , Cell Nucleolus/metabolism , Cell Proliferation/drug effects , Doxycycline/pharmacology , Gene Knockdown Techniques , HEK293 Cells , Humans , Immunoprecipitation , Kinetics , Methyltransferases/genetics , Methyltransferases/isolation & purification , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Protein Binding/drug effects , Protein Transport/drug effects , RNA/metabolism , RNA Interference/drug effects , RNA Processing, Post-Transcriptional/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of ResultsABSTRACT
Human NY-REN-21 is a C2H2 type multi-finger protein, with a SCAN domain in the N-terminal region and a predicted coil central region. It represents a putative ortholog of mouse ZFP38, a transcriptional factor that recognizes a bipartite DNA motif and is unable to form homodimers. As shown in this work, NY-REN-21 contains a SCAN domain able to form homodimers and a central region that behaves as an intrinsically disordered protein. The SCAN domain is found in 71 human proteins and its ability to form homo- and heterodimers widens the number of genes that are regulated by this group of transcription factors. NY-REN-21 interaction with SCAND1 was identified using the yeast two-hybrid system and confirmed using recombinant proteins. SCAND1 is a truncated SCAN box protein, lacking the zinc finger region and the NY-REN-21/SCAND1 heterodimer is asymmetric concerning the DNA binding region. This result indicates that NY-REN-21 can function either as a homodimer or as a heterodimer with SCAND1.
