Abstract
Abstract
Background: Given the importance of iron in human health, surprisingly there are many gaps in our knowledge of how cells can rapidly respond to changes in extracellular iron levels. There is a lack of research investigating cellular response to iron in a short time frame (hours). Much of the known rapid response to iron is facilitated through structured mRNA cis-regulatory elements, Iron-Responsive Elements (IRE). These can be bound by IRE-binding proteins when cellular iron levels are low, and influence protein production. All characterised IRE sequences have a high level of conservation in RNA secondary structure, but vary in primary sequence. Because of this, bioinformatic predictions of putative IRE sequences have been done. This research uses a combination of high-throughput screens and reporter gene analysis to examine a cellular response to iron after 6 h, aiming to test sequences predicted to form IREs, and identify genes with a post-transcriptional response to iron.
Results: Eleven previously predicted novel IREs from human genes were tested in cell culture for iron response. These putative IREs were cloned into the 5′ untranslated region (UTR) of a luciferase reporter gene, to detect if the putative IREs showed a strong post-transcriptional response to iron. The IRE from the high mobility group box 1 (HMGB1) gene promoted significantly greater reporter gene expression in high iron than with an iron chelator within 6 h of treatment. This indicated a rapid post-transcriptional response to iron.
Independently, to examine a genome-wide response to iron over 6 h, and indicate candidate genes for further analysis; pulsed stable isotope labelling by amino acids in cell culture (pSILAC) and RNA sequencing were performed. From this a transcriptome and proteome were obtained. As expected there was no significantly different levels of mRNAs between treatments. However, 21 proteins showed a strong post-transcriptional response to iron. Many of these proteins were involved directly in iron metabolism and/or, contained IRE-like sequences. Two proteins, eukaryotic initiation factor 5, and elongation factor 1A, showed a large increase of protein in high iron compared to the iron chelator (Log2 fold change = 4.2 and 3.9, respectively). Across the experiment there was more protein synthesis in cells treated with iron compared to cells treated with the iron chelator (Log2 change = 2).
Conclusions: The putative IRE sequence from HMGB1 showed a rapid post-transcriptional response in cells treated with iron. This indicates the putative IRE may function in HMGB1 expression. Further analysis directly looking at this in HMGB1 needs to be performed.
Several of the 21 proteins from the screen are good candidates for detailed analysis, two of these proteins are directly involved in translation and may be responsible for the difference in protein production in iron treated cells.