Coming to you LIVE from the 5th annual RNA Symposium: Processing RNA. Follow us on Twitter or the tag #umichrna!

Live blogger: Zoe Yeoh

Editor: Alyse Krausz

Recently, RNA has risen to the forefront of nucleic acid research due to its newly characterized, integral roles in cellular regulation. Many of the secrets in RNA regulation lie in its ability to form complex secondary and tertiary structures that relate to its function. However, many of these structure-function relationships are poorly characterized due to a lack of rigorous tools used to study them. Dr. Kevin Weeks and his group at University of North Carolina-Chapel Hill have developed “novel chemical microscopes that reveal quantitative structure and function interrelationships for RNA” to study these complex RNA structures and answer important biological questions.

Dr. Weeks’ group pioneered a method called SHAPE-MaP (2′-hydroxyl acylation analyzed by primer extension and mutational profiling). The development of this method was driven by the desire to have a straightforward way to identify RNA secondary structure and has now been used for almost a decade.

Some features of RNA secondary structure. (Source)

SHAPE-MaP is a technique that enables the large-scale identification of RNA regions that have some biological function. These biologically active, sequence-specific RNA regions are called functional motifs. Proteins that help mediate biological functions of RNA need access to these RNA functional motifs, so functional motifs are often found in flexible, non-base-paired regions of RNA. 

The SHAPE-MaP method labels nucleotides in these flexible regions with a SHAPE chemical probe through a process called covalent modification. Complementary DNA (cDNA) can then be synthesized using the covalently modified RNA as a template. These cDNA sequences hold information about the RNA structure because a non-complementary nucleotide is added on the cDNA where the template RNA was covalently modified. A method called massively parallel sequencing is then used to analyze multiple cDNAs at the same time. 

This process results in a reactivity profile, which ultimately characterizes RNA secondary structure. The more reactive an RNA sequence is, the more flexible; the less reactive, the more base-paired and structured. The reactivity is mapped onto the RNA sequence and analyzed for base-pairing probability. Finally, the secondary structure can be computationally modelled using the reactivity probabilities. 

In his talk, Dr. Weeks highlighted several lessons learned from using this method. SHAPE-MaP offers a direct readout for RNA secondary structure probing and is most effective using high-quality RNA models in vitro. Often, the accessibility of unstructured single-stranded RNA, rather than specific secondary structure patterns, correlates to RNA functionality and associations. Further, each functional RNA has its own ‘personality’, leading to a huge diversity in specific functions and associations with other biomolecules. 

The Weeks group used SHAPE-MaP to look at the HIV-1 genome, where they found RNA regions with low SHAPE reactivity, low Shannon entropy (a measure of disorder), and many structural motifs. Interestingly, regions with high Shannon entropy (or high disorder) appeared to be more prone to interactions with proteins. These metrics of low SHAPE reactivity and low Shannon entropy have been utilized by other groups and applied to other models such as Dengue virus, SARS-CoV-2, therapeutic RNAs, and long non-coding RNAs. 

SHAPE-MaP is useful for analyzing RNA structure in a local, in vitro setting, but Dr. Weeks wanted to understand the impacts of RNA structure on a cellular scale. In addition, he highlighted a notable limitation of SHAPE-MaP: it requires computational modelling to obtain secondary structure information. 

To address this limitation, the Weeks group developed a method called PAIR-MaP (pairing ascertained from interacting RNA strands measured by mutational profiling) to directly profile base-paired regions instead of relying on modelling. This method uses a type of chemical probing called dimethyl sulfate (DMS) under particular experimental conditions to modify base-paired nucleotides. Paired nucleotides are normally protected, but they also exist in equilibrium in an unpaired state. In the unpaired state, the nucleotides can be modified by DMS and detected using the MaP strategy. Impressively, the Weeks group used PAIR-MaP to predict every RNA pseudoknot in the E. coli genome (except for one).

But what about RNA tertiary structure? Is there a way to distinguish between heterogeneous RNA states present? Dr. Weeks illustrated a method called RING-MaP (RNA interaction groups measured by mutational profiling) that could answer both of these questions simultaneously. Where the previous approaches used ‘per nucleotide’ probing, RING-MaP used ‘correlative’ chemical probing. As an example of RING-MaP’s utility, Dr. Weeks explained that this method was used to look at the Adenine riboswitch, a well-characterized RNA molecule that has two states – an ‘on’, or Adenine-bound, state, and an ‘off’, or unbound state. Using RING-MaP, the group saw these two previously characterized states and could even determine a binding constant for the molecule to adenine. They could also deconvolute the percentage of ‘on’ versus ‘off’ RNA, and found that the ‘on’ conformation dominated.

RING-MaP method pipeline. (Source)

The latest method the Weeks group has developed aims to answer all of the previously posed questions in a single step. This method, called DANCE-MaP (Deconvolution and annotation of ribonucleic conformational ensembles measured by mutational profiling) combines aspects from all of the previously mentioned assays (SHAPE-MaP, PAIR-MaP, and RING-MaP) and was led by a graduate student in the Weeks lab, Sam Olsen (paper currently in preparation). The group was able to apply this method to an RNA called 7SK, a non-coding RNA which regulates transcription, and revealed exciting new insights into how transcription is regulated on the RNA structure level.

In summary, Dr. Weeks and his group have developed a series of exciting and powerful tools to answer questions about RNA secondary and tertiary structure, homogeneity, and functionality.

Photo by Lars Sahl (Source)

Dr. Weeks received his Ph.D. at Yale University in 1992 and went on to the University of Colorado as a Postdoctoral Fellow. He is a Kenan Distinguished Professor in the Department of Chemistry at the University of North Carolina – Chapel Hill, where he has been a faculty member since 1996.  He is also the co-founder of Ribometrix, a platform therapeutics company discovering small molecule drugs that target functional 3D RNA structures to treat human diseases.

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