Lab News

1/22/2021 – We discussed Yu et al.‘s recently published paper suggesting that RNA structural intermediates may be recognized by RNA processing machinery and that the transitional landscape of folding should be considered for regulation and function from the Lucks lab. Realistic molecular dynamic simulation of RNA molecules remains difficult, however, simulations can provide insight into the biology of RNA structure. Yu, et al.’s paper addresses some very tricky questions on co-transcriptional folding and how structures evolve during transcript to result in their final mature form. The R2D2 (reconstructing RNA dynamics from data) method uses experimental SHAPE data during impaired transcription to capture structures of progressively longer transcripts. The authors took this to the next step and performed molecular dynamic simulations on these structural stages. They delve deep into the maturation of the SRP RNA. The highlight I took from this paper is that key nucleotides that influence the final RNA structure may not be those that form a long hairpin, but the nucleotides that stabilize or destabilize intermediary structures. More work is needed to understand how the lessons from SRP apply to other RNAs and how to identify key nucleotides. In particular, I’d be interested in learning more about the conservation of nucleotides that participate in intermediary structures and whether variants in intermediary positions are associated with human disease.

Upcoming (1/29/2021) – First data presentation in the lab! Luke Hatfield will present an overview of his projects since he joined in early December.

1/15/2021 – We read Marinus et al.‘s publication on developing novel SHAPE reagents to probe cellular RNA structure and improve signal-to-noise ratio in structural data from the Incarnato lab. There are a variety of RNA secondary structure probes, but all have caveats. For example, dimethyl sulfate (DMS) is highly reactive and easily penetrates cell membranes, but it only reacts well with A and C nucleotides (and it is highly toxic!). Most sensitive chemical probes like 1M7 and NIA result in better structure information from all four bases, but do not easily penetrate cell membranes and require extensive effort to develop good signal from background noise. Marinus et al. address this problem by synthesizing additional reagents based on NIA. The best of the new reagents, 2-aminopyrimidine-3-carboxylic acid imidazolide (2A3), only differs from NIA by replacing a methyl group with an amino group. However, this subtle difference makes this molecule more reactive with RNA, dramatically improving the signal-to-noise ratio, and allows the molecule to penetrate cell membranes more effectively. I would have liked to see the comparison with 5NIA, which I have used in lab and can permeate some types of mammalian cells. However, I think an additional reagent that improves RNA secondary structure experimental data is extremely useful and hope to test it out soon!

Upcoming (1/22/2021) – We will discuss Yu et al.‘s publication suggesting that RNA structural intermediates may be recognized by RNA processing machinery and that the transitional landscape of folding should be considered for regulation and function. This paper was recently published from the Lucks lab.

1/8/2021 – For our first meeting of 2021, we discussed Liu, et al.’s 2021 paper on the nuclear structurome from the Ding lab. The authors used Arabidopsis seedlings (dunked in RNA secondary structure chemical probing reagents) to develop RNA structures on cytoplasmic and nuclear RNAs. At the core of this paper is the idea that in vivo and in vitro RNA structures differ significantly and, by extension, that precursor mRNA and mature mRNA also have dramatically different structures. This would imply that the structures of mature mRNAs are not accurate representations for understanding RNA binding protein recognition, particularly in the context of splicing and alternative polyadenylation. The authors also support the ability of SHAPE reagents (NAI) to detect protein binding by comparing the reactivity of “deproteinized” RNAs to RNA protein complexes. By comparing the reactivity of the nuclear and cytoplasmic RNAs, Liu et al. detect a 2-nucleotide “structural motif” of unstructured RNA at the -1 and -2 positions around the 5′ splice site (within the exon). This “motif” corresponds with better recognition of the splice site and, in their experiments, are the only nucleotides required to be unpaired within the entire U1 snRNP recognition motif (spans -3 to +6). They did not detect a clear structural signal at the 3′ splice site, but did see an increase in single-stranded signal around the branch point. Likewise, strong polyadenylation signals have an overall high reactivity, suggested unpaired nucleotides. Is the difference between nuclear and cytoplasmic RNA structure due to protein binding differences between the two compartments? What the mechanism underlying dramatic structural changes? Is this an active process or inherent in the splicing transition where two exons form newly conjoined sequences that rearrange? I think the idea of RNAs with difference nuclear and cytoplasmic structures is intriguing. I’d like to see whether this concept and the 5′ splice site “structural motif” hold up humans.

Upcoming (1/15/2021) – We will discuss Marinus et al.‘s publication on novel SHAPE reagents with new abilities from the Incarnato lab!

12/18/2020 – We discussed Mi Seul, et al.’s paper on AGO3 structure and activity (2017) from the Nakanishi lab, as well as a secondary paper on AGO3 activity (2020) with tyRNAs (tiny RNAs that are 14-17 nts long). My favorite part of the 2017 paper is the beautiful crystal structure of AGO3 + RNA the authors present. It is available on the pdb under the id 5VM9. In addition to their structural studies the authors also show compelling biochemical experiments demonstrating that the AGO3/microRNA complex is capable of cleaving target mRNAs under specific circumstances. Although AGO2 easily binds a wide variety of microRNAs and directs mRNA cleavage based on the microRNA/mRNA interaction, AGO3 was previously thought to be inactive. Any residual activity in AGO3 preparations in biochemical assays was thought to come from contaminating AGO2. By preparing and purifying AGO3 from insect cells the authors clearly show that AGO3 in complex with specific microRNAs can cleave mRNAs. In their most recent publication, Mi Seul, et al., expand their analysis and show that AGO3’s cleavage ability is influenced by the size of the small guide RNA. In comparison to AGO2, AGO3 poorly cleaves mRNAs when it is complexed with a typical microRNA (21-23nt long). However, AGO3 outperforms AGO2 when it is complexed with small guide RNAs (14 nt long). Interestingly the sequence of these cleavage inducing tiny RNAs (cityRNAs) is more important for cleavage than traditional microRNA guided cleavage by AGO2. It remains difficult to determine which mRNAs AGO3 activity might be relevant for, as these papers still only test a handful of microRNAs and similar cityRNAs. We look forward to learning more about the functions of other, less known members of the argonaute family.

Upcoming (12/31/2020): We are reading Bubenik, et al.’s recent paper on deriving RNA structure from low abundance transcripts (i.e. pre-cursor mRNAs) from the Berglund lab.

There is so much amazing RNA research going on! We have a weekly journal club to talk about the latest publications. Here are the papers that we have been reading:

Upcoming 12/11/20: We are reading Rafael de Cesaris Araujo Tavares, et al.’s paper from Anna Marie Pyle’s lab about RNA structure across SAR-CoV2. This paper was recently published in the Journal of Virology – The global and local distribution of RNA structure throughout the SARS-CoV-2 genome.

12/4/20: We read Meredith Corley, et al.’s paper from Gene Yeo’s lab describing the fSHAPE protocol and its relevance. This paper was recently published in Molecular Cell – Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.

One interesting result we noticed in this paper was that RNA binding proteins generally interact with well structured regions (low Shannon entropy), but are more likely to directly contact accessible nucleotides within those structured regions. A question we would have liked to see more about in the discussion is how transcriptome-wide SHAPE protocols performed in the presence of proteins are influenced by protein protections and whether in-cell structure data is inaccurate or if eCLIP is necessary to see protein protection in standard SHAPE protocols.