Biology subjects

Kaczynski, T. J.

Publications and source records attributed to Kaczynski, T. J..

2 recordsLinked to original sources

bioRxiv2022

Exploring the lncRNA localization landscape within the retinal pigment epithelium under normal and stress conditions

Long noncoding RNAs (lncRNAs) are emerging as a class of genes whose importance has yet to be fully realized. It is becoming clear that the primary function of lncRNAs is to regulate gene expression, and they do so through a variety of mechanisms that are critically tied to their subcellular localization. Although most lncRNAs are poorly understood, mapping lncRNA subcellular localization can provide a foundation for understanding these mechanisms. Here, we present an initial step toward uncovering the localization landscape of lncRNAs in the human retinal pigmented epithelium (RPE) using high throughput RNA-Sequencing (RNA-Seq). To do this, we differentiated human induced pluripotent stem cells (iPSCs) into RPE, isolated RNA from nuclear and cytoplasmic fractions, and performed RNA-Seq on both. Furthermore, we investigated lncRNA localization changes that occur in response to oxidative stress. We discovered that, under normal conditions, most lncRNAs (76%) are seen in both the nucleus and the cytoplasm to a similar degree, but of the transcripts that are highly enriched in one compartment, more are nuclear (18.6%) than cytoplasmic (5.6%). Interestingly, under oxidative stress conditions, we observed an increase in lncRNA localization in both nuclear (23.5%) and cytoplasmic (9.7%) fractions. In addition, we found that nuclear localization was partially attributable to the presence of previously described nuclear retention motifs, while adenosine to inosine (A-to-I) RNA editing appeared to play a very minimal role. Our findings regarding lncRNA localization in the RPE provide two avenues for future research: 1) how lncRNAs function in the RPE, and 2) how one environmental factor, in isolation, may play a role in retinal disease pathogenesis.

genetics↗

bioRxiv2021

Oxidative stress alters transcript localization of disease-causing genes in the retinal pigment epithelium

Nuclear retention is a mechanism whereby RNA transcripts are held in the nucleus to maintain a proper nuclear-to-cytoplasmic balance or as a stockpile for use in responding to stimuli. Many mechanisms are employed to determine whether transcripts are retained or exported to the cytoplasm, though the extent to which tissue- or cell-type, stressors, or disease pathogenesis affect this process remains unclear. As the most biochemically active tissue in the body, the retina must mitigate endogenous and exogenous stressors to maintain cell health and tissue function. Oxidative stress, believed to contribute to the pathogenesis, or progression, of age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs), is produced both internally from biochemical processes, as well as externally from environmental insult. To evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), we performed poly-A RNA sequencing on nuclear and cytoplasmic fractions from induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide, as well as untreated controls. Under normal conditions, the number of mRNA transcripts retained in the nucleus exceeded that found in studies of other tissues. Further, the nuclear-to-cytoplasmic ratio of transcripts is altered following oxidative stress, as is the retention of genes associated with AMD, IRDs, and those important for RPE physiology. These results provide a retention catalog of all expressed mRNA in iPSC-RPE under normal conditions and after exposure to hydrogen peroxide, offering insight into one of the potential roles oxidative stress plays in the progression of visual disorders.

genetics↗