Live Blogger: Liz Tidwell
Editor: Henry Ertl
Because they do not encode instructions for protein products, the role of non-coding RNA in biological processes was overlooked for decades. With the discovery of regulatory RNA, such as small RNA in bacteria (sRNA), noncoding RNAs (ncRNA) are starting to be appreciated for their role in gene regulation. During her talk at the University of Michigan’s 2022 RNA Symposium, Dr. Gigi Storz presented compelling data to extend the limited definition of sRNA: sRNA within translated regions, some of which may be coding something after all.
The existing paradigm of sRNA is three-fold:
- sRNAs are encoded as distinct transcripts in intergenic, noncoding regions
- sRNAs regulate expression by base pairing to the 5’ UTR of messenger RNA (mRNA)
- sRNA have one function
In today’s seminar, Dr. Storz presented evidence to challenge each of these assumptions, indicated below by the strikethrough headings under each tenet of the old paradigm.
sRNAs are encoded as distinct transcriptions in intergenic noncoding, regions
Deep sequencing data of the E. coli genome revealed sRNA sequences that surprisingly overlap coding sequences. To understand possible functions of sRNAs that overlap coding sequences, Dr. Storz’s group focused on an sRNA integral to the cutC gene, which was thought to be responsible for the tolerance of copper in bacteria. Removing the sRNA but maintaining protein expression made bacteria sensitive to copper. When the sRNA was overproduced, copper tolerance was rescued. This suggests that the sRNA caused the phenotype, rather than the cutC protein! Using a combination of RNA sequencing and crosslinking (RIL-seq), Dr. Storz’s group also identified a second internal sRNA, FtsO. This sRNA, located in the ftsI coding sequence, doesn’t regulate mRNA. Instead, it specifically binds to the RybB sRNA, acting as a sRNA sponge. These findings show that sRNAs can be found outside of the untranslated regions and have key roles in bacterial gene regulation.
sRNAs target 5’ UTR of messenger RNA (mRNA) and regulate expression by base pairing
The identification of the FtsO sRNA contradicts the idea that sRNAs only regulate protein expression by mRNA binding in the 5′ UTR of mRNA. With further investigation, the Storz group identified an sRNA that regulates protein activity rather than protein expression. For example, the ArcZ sRNA regulates the LigA gene, which codes for bacterial DNA ligase. When ArcZ is overexpressed, there are no changes in the level of DNA ligase protein or LigA mRNA. However, when you measure the rate of DNA ligation, there is an increase in ligated DNA. Since ArcZ binds to the mRNA downstream of the NAD+ domain of the encoded DNA ligase, Dr. Storz hypothesizes that this sRNA slows down translation, increasing the probability of the protein interacting with the cofactor. This hypothesis was further validated by experiments that showed that the ArcZ sRNA is upregulated under starvation conditions, when bacteria most need to conserve cellular resources.
sRNA have one function
One of the first regulatory RNAs, Spot 42, was identified in the 1970s and is known to regulate a protein called cyclic adenosine monophosphate receptor protein (CRP), which is involved in alternate pathways of carbon metabolism in E. coli. CRP increases the transcription of genes involved in the uptake and use of non-glucose carbon sources in low glucose environments. Initially, Spot 42 was understood to induce downregulation of mRNA via base pairing in the galactose metabolism operon, which reduces the use of galactose as a carbon source when glucose is present. In 2022, the Storz lab discovered that Spot 42 also encodes a small, 14-amino acid protein. Through ribosome profiling, protein pulldown assays, and a series of sequence modifications, they determined that the encoded protein SpfP and Spot 42 regulate CRP together. In addition to the effects of Spot 42’s base pairing, SpfP binds to CRP, which blocks its ability to upregulate the transcription of alternate carbon source proteins.
However, the most interesting finding was that SpfP and Spot 42 do not affect CRP during all environmental conditions. This is because SpfP is only slightly expressed when E. coli is grown under normal temperatures (30°C). Under higher temperatures, SpfP expressed at much higher levels. This finding suggests that the activity of Spot 42 wanes at high temperatures (40°C), but the regulation of carbon metabolism proteins is maintained by its encoded protein!
Moving forward with a new paradigm
Altogether, these findings show the wonderful complexity of gene regulation even in organisms that have been studied for so long. Dr. Storz started her talk by challenging the audience to consider the potential of the production of alternative proteins from all RNA, including RNAs formerly considered to be noncoding. It is clear from Dr. Storz’s work that there is much left to discover about genomic regulation encoded in RNA. sRNA are much more varied and complex than previously thought. Her work will continue to probe the question of how organisms use RNA to respond to environmental changes, using the new paradigm of noncoding RNAs she laid out for us today:
- sRNA are encoded within coding sequences as well as intergenic regions of the genome.
- sRNA use base pairing with one or more RNA to regulate processes in response to environmental or cellular factors. The exact method of regulation can vary widely.
- sRNA can encode small proteins and can have multiple functions.
Dr. Gisela Storz’s research interest in genetic regulation began during her Ph.D. at the University of California Berkeley where she investigated oxidative stress responses in bacteria. Her post-doctoral research branched out into plant biology, studying how Arabidopsis regulates light intake at Harvard Medical School. However, her focus never left stress responses. Currently, she works at the National Institute of Child Health and Human Development Division of Molecular and Cellular Biology. Through her research into how yeast and bacteria respond to stress, her group discovered a master transcription regulator in bacteria, and a nuclear localization regulator in yeast, both of which reduce the toxic effects of oxidative stress. Serendipitously, they also discovered that those master regulatory genes code for prototypical sRNA. This discovery pivoted her work to understand how bacterial sRNAs are encoded, how these sRNAs regulate gene expression and the impact of these regulation systems on the evolution of bacteria and yeast. Her work has earned her the title as NIH Distinguished Investigator and a wide range of awards and honors.