Abstract
Purpose :
The αA-crystallin gene is highly expressed in the developing ocular lens. Along with αB-crystallin, the two alpha-crystallins make up about 30% of the gene products in the lens. This investigation focused on gaining insight into a molecular mechanism for sustaining high transcript levels of Cryaa in the developing ocular lens.
Methods :
As a part of our investigations on the regulation of gene activities in the developing lens, we have been working with the transcriptomic characterization of manually isolated single fiber cells. We have developed procedures for isolating single fiber cells from the postnatal day two (PND02) mouse lens. We used various technologies to generate cDNA libraries from RNA isolated from single fiber cells and assessed the transcription levels obtained through Next Generation single-cell RNA sequencing. We further probed with multiple RT-PCR technologies.
Results :
Single-cell cDNA libraries made with template switching technologies generate higher amounts of transcripts, including more protein-coding genes (up to about 20% more), with smaller but modest increases in other transcripts, including the long noncoding (lnc) RNAs. We make the following observations: 1. The ENSEMBL genome browser indicated that the antisense Lnc covers all of the αA-crystallin gene locus. 2. We generated libraries from representative fiber cells isolated from the equatorial, cortical, and nuclear regions. In many fiber cells, we found that this Lnc was one of the top 10 gene products, including several crystallin genes. 3. The expression of the lnc RNA is well expressed in all the fiber cells analyzed, and more importantly, it represents more than 20-30% of the transcripts made from the aA-crystallin locus. 4. The noncoding lnc RNA and the Cryaa transcripts are expressed in the same cell. 5. Finally, RT-PCR analyses indicate predominant expression of the 3’ transcripts of the lnc RNA in the ocular lens.
Conclusions :
The natural antisense transcript(s) made from the entire length of the Cryaa gene may suggest multiple modes of regulation. However, the expression of 3'-antisense transcripts may indicate their involvement in sustaining increased levels of Cryaa transcription.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.