The bZIP-like motif of hnRNP C directs the nuclear accumulation of pre-mRNA and lethality in yeast

• Published in: Journal of molecular biology 2001-01, Vol.305 (4), p.829-838
• Main Authors: Tan, Jia-huai, Kajiwara, Yuji, Shahied, Lillian, Li, Jun, McAfee, James G, LeStourgeon, Wallace M
• Format: Article
• Language: English
• Subjects: AC protein; Active Transport, Cell Nucleus; Amino Acid Motifs; C protein; Cell Nucleus - metabolism; chaperonin; chaperonins; Circular Dichroism; Fluorescence; Genes, Lethal - genetics; Heterogeneous-Nuclear Ribonucleoprotein Group C; Heterogeneous-Nuclear Ribonucleoproteins; hnRNA; hnRNP; Humans; In Situ Hybridization; leucine zipper; Leucine Zippers; Protein Binding - drug effects; Protein Structure, Quaternary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; ribonucleoprotein; Ribonucleoproteins - chemistry; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; RNA Precursors - genetics; RNA Precursors - metabolism; RNA, Fungal - genetics; RNA, Fungal - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-binding; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Salts - pharmacology; Sequence Deletion - genetics; splicing factors; Thermodynamics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.4332

The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo. Based on these and other findings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors. We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae. Through a series of fluorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA. Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-affinity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214). Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein’s salt-resistant RNA binding mode. Taken together, these findings further distinguish the bZIP-like domain as the major determinant of C protein’s high-affinity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity. Finally, these findings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast. Lethality is likely due to the absence in yeast of specific mechanisms for the removal of human C protein from nascent transcripts.