Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human β-Myosin Heavy Chain Gene

Abstract

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

Characterization of cellular nucleic acid binding protein fromXenopus laevis: Expression in all three germ layers during early development

“…CNBP has also been shown to interact with a repressor domain in the human cardiac ␤-myosin heavy chain (␤-MHC) gene (Flink and Morkin, 1995a). The human CNBP gene encodes two alternatively processed isoforms, ␣ and ␤ (Flink and Morkin, 1995a).…”

“…The human CNBP gene encodes two alternatively processed isoforms, ␣ and ␤ (Flink and Morkin, 1995a). CNBP␣ may function as a negative transcriptional regulator by binding to the promoters of target genes containing guanine-rich sequences related to the SRE that are found within the human β-MHC promoter (Rajavashisth et al, 1989;Flink and Morkin, 1995a).…”

Novel CNBP‐ and La‐based translation control systems for mammalian cells

“…CNBP has been reported to be a single-stranded nucleic acid-binding protein involved in the production of the lowdensity lipoprotein (Rajavashisth et al, 1989), regulation of the c-myc oncogene (Michelotti et al, 1995) and the expression of the human ␤-myosin heavy chain (Flink and Morkin, 1995a). The predominant localization of CNBP in the cytoplasm or the endoplasmic reticulum is consistent with its preferential binding to specific RNA sequences (Warden et al, 1994).…”

“…pSS107: The human cellular nucleic acid binding protein (CNBP) was excised from pSG5-CNBP (Flink and Morkin, 1995a;1995b; by StuI/AflIII, polished with Pfu polymerase and ligated into the SmaI site of pBluescriptII KS − .…”

“…The vectors for expression of the TOP binding proteins CNBP (Flink and Morkin, 1995a;1995b) and La (van Venrooij et al, 1993) are isogenic and contain a tricistronic P hCMV*−1 -driven expression cassette encoding in the first cistron the single-chain urokinase-type plasminogen activator (u-PA LMW ; Langer et al, 1995), either CNBP (pSS124) or La (pSS125) in the second and the zeocin resistance marker in the terminal cistron (pSS124: P hCMV*−1 -u-PA LMW -IRES-CNBP-CITE*-zeo-pA; pSS125: P hCMV*−1 -u-PA LMW -IRES-La-CITE*-zeo-pA) (Fig. 1B).…”