Live blogger: Varsha Shankar

Editors: Sadie Gugel and Jennifer Baker

This piece was written live during the 7th annual RNA Symposium, “From Molecules to Medicines,” hosted by the University of Michigan’s Center for RNA Biomedicine. Follow MiSciWriters’ coverage of this event on Twitter with the hashtag #umichrna.

You may recall learning in high school biology that ribosomes are the smallest organelle. Despite their miniscule size, these organelles are one of the most critical – that’s why they, unlike some organelles, are present in both eukaryotes and prokaryotes. The site of protein synthesis in the cell, ribosomes are responsible for building proteins that dictate our bodily metabolic activity, and ultimately, who we are. 

Ribosomes recognize the messenger RNAs (mRNAs) to be translated via binding sites, where the mRNA and ribosome are complementary and can thus initiate the translation process. Dr. Joseph Wedekind elaborates on the structure and function of these binding site sequences in mRNA through his work on riboswitches. 

Riboswitches, defined as RNA structures that function in gene regulation, are found in the 5’ leader sequences of bacterial mRNAs. These riboswitches bind to certain molecules, thereby regulating translation by changing the conformation of the ribosome binding site, and thus affecting the ability of the ribosome to bind. There are many types of riboswitches with a range of functions, from splicing control to translation control. One specific type of riboswitch that Dr. Wedekind and his lab focus on is preQ1-sensing riboswitches, which bind pre-queuosine 1, thus regulating the genes involved in synthesis of the queuosine, a nucleoside present in tRNA.

Dr. Wedekind and his group have previously determined the structures and functions of different preQ1 riboswitches, and these discoveries along with other research indicate that preQ1 riboswitches bury the SDS ribosomal binding site to prevent translation. Dr. Wedekind and his lab’s research now hypothesize something different due to the discovery of a new class of riboswitches: the possibility that for some riboswitches, this SDS binding site does not have to be buried. There may be another process that is the internal link to gene regulation in some cases. 

The Shine-Dalgarno sequence (SDS) is a ribosomal binding site necessary for translation to occur. In the traditional model of the preQ1 riboswitch, the SDS on the RNA being regulated binds with the anti-SDS of the riboswitch. This riboswitch forms an SDS-anti-SDS conformation that sequesters the SDS, thus stopping translation when ligands bind. However, Dr. Wedekind’s lab has found exceptions to this rule: the SDS may not interact with the anti-SDS in certain preQ1 riboswitch types, and moreover, the SDS may not have to be buried for regulation to occur.

So if the SDS does not have to interact with the anti-SDS region of the riboswitch, how is the riboswitch still able to regulate translation? First, a bit of background about the various types of preQ1 riboswitches. preQ1 is the only soluble precursor to queuosine, a component of tRNA. preQ1 riboswitches form pseudoknots to regulate translation by impacting the binding sites via ribosomal interactions. There are three classes of preQ1 riboswitches that vary based on the type of pseudoknot they form. Class 1 preQ1 riboswitches include three subtypes with slight differences in the SDS binding method, but overall, the ligand binds to the alpha binding site for gene regulation to occur. For Class 2, the ligand binds to the beta binding site, and for class 3, the ligand binds to the gamma binding site.

Dr. Wedekind and his lab conducted an experiment where they tested whether the absence of the preQ1 riboswitch would impact expression of a gene, which encoded GFP so they could visualize when it was expressed. For Class 2 preQ1 riboswitches, they found that when the preQ1 riboswitch was absent, GFP production was impaired, indicating this class uses the traditional pathway of SDS-anti-SDS interaction and SDS burial for gene regulation. For Class 3, they found that preQ1 allows for docking of SDS with anti-SDS. This indicates that SDS and anti-SDS are not consistently paired, but SDS still had to be buried for gene regulation to occur, following the traditional model to a slightly lesser extent than Class 2 riboswitches. 

But for Class 1 preQ1 riboswitches, the result was interesting. For Class 2 and 3 preQ1 riboswitches, only one ligand has to bind to the binding site for the riboswitch to execute its regulatory role. However, gene regulation by the Class 1 preQ1 riboswitch requires two ligands in the binding pocket. Further, Class 1 type 1 preQ1 riboswitches do not have to sequester SDS. How did they figure this out? The lab removed the SDS from the expression platform and still saw significant levels of gene regulation even though SDS did not overlap with anti-SDS. The type 1 Class 1 preQ1 riboswitch is the exception in the rule; while the other classes of preQ1 riboswitches require SDS-anti-SDS interaction and SDS burial for gene regulation, the pseudoknot is the internal link to gene regulation for this riboswitch.

In summary, Dr. Wedekind and his lab discovered that Class 2 and 3 preQ1 riboswitches require total or partial SDS-anti-SDS pairing, leading to SDS sequestration, for gene regulation to occur. For Class 1 forming the pseudoknot confirmation is more essential for gene regulation than SDS-anti-SDS pairing. Specifically, Class 1 Type 1 preQ1 riboswitches have no SDS-anti-SDS pairing and still employ gene regulation. 

What does uncovering a new riboswitch mechanism of gene regulation mean in the bigger picture? This is an important finding, as SDS sequestration was previously thought to be the canonical mechanism for gene regulation by these riboswitches. The newly found dispensability of SDS burial for gene regulation in some riboswitches may mean that other riboswitches have been missed since this mechanism had not been previously considered.

Dr. Joseph Wedekind is a Professor in the Department of Biochemistry and Biophysics at the University of Rochester. He earned his B.S. in Biochemistry at University of California – Riverside, completed a Ph.D. in Biochemistry at The University of Wisconsin – Madison, and completed his postdoctoral training in RNA biophysics at Stanford University. Dr. Wedekind’s research interests include the structure and function of non-coding RNAs, including various types of riboswitches and their functions. More information about Dr. Wedekind and his lab can be found here.

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