Identification of a promoter for the human C1Q-tumor necrosis factor-related protein-5 gene associated with late-onset retinal degeneration.
OBJECTIVE
The Complement-1q tumor necrosis factor-related protein 5 (C1QTNF5/CTRP5) gene is located in the 3' untranslated region of the Membrane Frizzled Related Protein (MFRP) gene, and these two genes are reported to be dicistronic. The authors examined the 5' upstream sequence of CTRP5 for the presence of a promoter regulating the expression of this gene.


METHODS
The sequence upstream of the translational start site of human CTRP5 (hCTRP5) was analyzed by Promoter Inspector software. A series of plasmids containing segments of hCTRP5 putative promoter sequence (-29 bp to -3.6 kb) upstream of the luciferase gene were generated. Cells were transiently transfected with these plasmids, and luciferase activity was measured. 5' RACE analysis was performed to determine the functional transcription start site. V5 tagged-pig CTRP5 (pCTRP5) gene, cloned downstream of the hCTRP5 putative promoter, was expressed in a human retinal cell line (ARPE-19) and a Chinese hamster ovary cell line (CHO-K1) to study the functionality of the putative promoter.


RESULTS
Bioinformatic analysis identified a putative promoter region between nt -1322 and +1 sequence of hCTRP5. 5' RACE analysis revealed the presence of the transcriptional start site (TSS) at 62 bp upstream of the start codon in the CTRP5. The 1.3-kb sequence of the hCTRP5 predicted promoter produced higher levels of luciferase activity, indicating the strength of the cloned CTRP5 promoter. The promoter sequence between nt -1322 bp to -29 bp upstream of the first ATG of CTRP5 was found to be essential for this promoter activity. The predicted hCTRP5 promoter was found to control the expression of V5-tagged pCTRP5 and nuclear GFP, indicating that the promoter was functional.


CONCLUSIONS
This study revealed the presence of a functional promoter for the CTRP5 gene located 5' of its start site. Understanding the regulation of CTRP5 gene transcription may provide insights into the possible role of CTRP5 in the retina and the pathology underlying late-onset retinal degeneration caused by mutations in this gene. In addition, these studies will determine whether CTRP5 and MFRP are functionally dicistronic.
Chavali
Venkata R M
VR
Department of Ophthalmology, University of California San Diego, La Jolla, California, USA.
Sommer
Jeffrey R
JR
Petters
Robert M
RM
Ayyagari
Radha
R
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003094:Collagen / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012162:Retinal Degeneration / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000099274:Frizzled-Related Proteins
D020413:3' Untranslated Regions; D003094:Collagen; D008565:Membrane Proteins; C487851:C1QTNF5 protein, human; C490110:MFRP protein, human; D000099274:Frizzled-Related Proteins