PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.
Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.
Manoharlal
Raman
R
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Gorantala
Jyotsna
J
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Sharma
Monika
M
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Sanglard
Dominique
D
Institute of Microbiology, University Hospital Lausanne, Lausanne CH-1011, Switzerland.
Prasad
Rajendra
R
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
D020413:3' Untranslated Regions; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D002176:Candida albicans / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002180:Candidiasis, Oral / Q000382:microbiology; D003001:Cloning, Molecular; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006720:Homozygote; D006801:Humans; D019656:Loss of Heterozygosity; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D000073718:Pancreatitis-Associated Proteins; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D020413:3' Untranslated Regions; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012331:RNA, Fungal; D012333:RNA, Messenger; D011061:Poly A; C548702:PAP1 protein, Candida albicans; D011062:Polynucleotide Adenylyltransferase; D001616:beta-Galactosidase