Characterization Of An Antisense RNA Of LAC1 In Cryptococcus Neoformans

Cryptococcus neoformans has emerged as a major fungal pathogen in immuno-compromised individuals such as patients with AIDS, organ transplant recipients etc. As a killer, C. neoformans has three main virulence factors:a capsule, laccase and the ability to grow at human body temperature. Laccase is one of the best established virulence factors for this human pathogen, and is expressed as a cell wall associated enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, LAC1 and LAC2, are present in the fungus, of which the first one is expressed for the majority of the enzymatic activity under glucose starvation conditions.Natural antisense transcripts (NATs) are endogenous RNA molecules containing sequences that are complementary to other transcripts. They belong to non-coding RNA family which is an important regulator of gene expression. NATs can be divided into cis-NATs, which are transcribed from opposing DNA strands at the same genomic locus, and trans-NATs, which are transcribed from separate loci. In eukaryotes, mainly mammal cell, cis-encoded antisense RNAs are the main type, but in prokaryotes, most of the antisense RNAs discovered are trans-encoded. At present, however, little is known about antisense RNAs encoded in fungi.In our work, we found that the antisense transcript of LAC1 (ALAC1) really existed. By now,1871 bp of its length was obtained, and which displayed perfect sequence complementarity to SLAC1 but without poly (A)-tail and had a certain kind of modification in the 3'-end. Then in quantitative RT-PCR assays, the transcripts of sense LAC1 (SLAC1) and ALAC1 were positively co-related, regardless of various mutants and conditions, and the transcriptional level of ALAC1 was always lower than SLAC1. Finally, over-expression of ALAC1 in trans, however, repressed the transcription of SLAC1, which indicated that there was a balance between the transcription of SLAC1 and ALAC1, and the natural antisense LAC1 possibly affected SLAC1 in cis, which does not have an interfering effect on the LAC1 mRNA.