Functional analyses of ζ-crystallin binding to the 3'-UTR of glutaminase (GA) mRNA

Chronic metabolic acidosis is associated with increased expression of renal glutaminase (GA) that results from selective stabilization of the GA mRNA. LLC-PK1- FBPase+ cells, a pH-responsive porcine kidney cell line, express four distinct GA mRNAs, that are ~2.5, 3.5, 4.5, and 5.0 kb in length. RNase H mapping indicated that three of the GA mRNAs are generated by the use of alternative polyadenylation sites and they are homologs of the rat kidney glutaminase (KGA) mRNA, while the 4.5 kb GA mRNA contains a different COOH-terminal coding and 3'-untranslated sequence. PCR cloning and sequencing confirmed that the 4.5 kb GA mRNA is the homolog of the human GA mRNA. Sequence analysis established that the 3'-untranslated regions (3'-UTR) of the 4.5 kb GA mRNA contains the 8-base adenylate-uridylate (AU) sequence that functions as a pH-responsive element (pHRE) The pHRE binds (ζ-crystallin /NADPH:quinone reductase (ζ-crystallin) with high affinity and specificity. Analysis of the influence of ζ-crystallin abundance on the GA mRNA level and stability was performed in vivo. LLC-PK1-FBPase+ cells that stably express a β-globin-glutaminase (βG-GA) construct were used to generate stable cell lines that over-express mouse ζ-crystallin. Western analysis established that a 4-fold increase in ζ-crystallin expression was obtained. An adenovirus was also used to over-express mouse ζ-crystallin. This approach produced 100-fold more mouse ζ-crystallin than the endogenous protein. An adenovirus that expresses a short interfering RNA (siRNA) reduced the expression of (ζ-crystallin by 90% compared to the endogenous protein in uninfected or in control cells. GA mRNA level and stability were determined with Northern blot and half-life analyses. No significant changes in the levels or stability of βG-GA mRNA were observed as a result of the increased or reduced abundance of ζ-crystallin. Thus, changes in (ζ-crystallin levels are not sufficient to affect the turnover or the pH-responsive stabilization of the GA mRNA. However, the possibility that events promoting modifications of (ζ-crystallin may affect its interaction with the GA mRNA or with other factors that affect its stability cannot be excluded. Various kidney cells express multiple isoforms of AUF1, an AU-binding protein that enhances mRNA turnover. RNA gel-shift assays demonstrated that the recombinant p40 isoform of AUF1 binds to the pHRE with high affinity and specificity. Thus, AUF1 may mediate the rapid turnover of the GA mRNA.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biochemistry and Molecular Biology

Grantor dc:publisher

Colorado State University. Libraries

Year dc:date.issued

2006

Authors dc:creator

• Ibrahim, Hend M. M., author
• Curthoys, Norman P., advisor
• Bamburg, James R., committee member
• Laybourn, Paul, committee member
• Stargell, Laurie A., committee member
• Mykles, Donald L., committee member

• biochemistry

Statement dc:rights

• Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.

Language dc:language.iso

eng, English

Identifier URI

https://doi.org/10.25675/3.026446

OAI identifier oai:identifier

oai:mountainscholar.org:10217/243726