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Higher-Order Organization Principles of Pre-translational mRNPs

Mihir Metkar, Hakan Ozadam, Bryan R. Lajoie, Maxim Imakaev, Leonid A. Mirny, Job Dekker, Melissa J. Moore

Preprint posted on March 08, 2018 https://doi.org/10.1101/278747

It’s all about folds and RIPPLes: principles for higher order organization of pre-translational mRNPs

Selected by Carmen Adriaens

It’s all about folds and RIPPLes: principles for higher order organization of pre-translational mRNPs

 

The idea:

A new technique named RIPPLiT combines RNA immunoprecipitation with proximity ligation to determine folding of RNAs within macromolecular messenger ribonucleoprotein (mRNP) particles.

 

What is this paper about?

In recent years, it has become increasingly clear that the 3D conformation of DNA and the correct folding of chromatin into contact frequency-determined topologically associating domains (TADs) is tremendously important for cellular identity and function. To study mRNA architecture and compaction in conjunction with their protein binding partners, in this work the authors apply the concept of contact frequency (here: chimeric junction frequency) and 3D conformation of macromolecules to develop a technique they name RNA ImmunoPrecipitation and Proximity Ligation in Tandem (RIPPLiT). With this method, they investigate the architecture of RNA within stable mRNPs, allowing to study the biogenesis, stability, folding and compaction of transcripts inside ubiquitous megadalton particles that protect and shuttle the RNA prior to translation.

The authors use this new technique to reveal the secondary structure of pre-translational RNA complexes through pulldown in tandem of two Exon Junction Complex (EJC) components. By means of proof-of-concept, they determine inter- and intramolecular junctions in ribosomal RNA. Because [as the authors put it] “the rules governing RNA polymerase II (Pol II) transcript packaging remain largely undefined”, they analyze the structure of Pol II transcripts in these complexes. Unlike previous cross-linking based studies, they find that Pol II transcripts, in their dataset, do not exhibit significant intermolecular contacts. In addition, they find that, independent of the length of the transcript, messenger RNA folding is relatively non-specifically distributed throughout the mRNP structural scaffold, resulting in densely packed, but flexible rods, rather than highly structured particles.

In short, they provide the first insights into how mRNAs are packaged in conjunction with their protein interactors, which are proposed to both protect them from degradation and shuttle them to the correct destination after their biogenesis.

 

My opinion on this preprint:

I like the fact that the authors apply a well-established concept – that of the importance of 3D architecture of macromolecules and complexes – to the study of (m)RNPs. They can now apply this technique to find the conformation of flexible RNA molecules within the protein particles that dictate their fate (e.g. translation, degradation, localization etc.), rather than to have to depend mainly on computational modeling. Furthermore, a major advantage is that unlike most other techniques for studying conformation of RNA, with RIPPLiT, they overcome the requirement for direct base-pairing of the RNA molecules to uncover spatial proximity.

It may be possible that the authors of this preprint detect very little intermolecular mRNA contacts because of the specific design of the technique (i.e. the use of Harringtonine to halt translation prior to protein pulldown), and the particular focus on the EJC and pre-translational complexes, rather than necessarily finding a global rule for mRNP biology. For instance, in another recent preprint (Morf et al., bioRxiv, 2017, https://doi.org/10.1101/196147), abundant spatial proximities are described for different RNAs in distinct nuclear particles, and it would be interesting to integrate data from these two techniques and others to ultimately obtain an integrative view on mRNP/RNA particle constitution in the cell.

Further implementation of RIPPLiT with other proteins in various cellular compartments will help to understand how RNAs interact within themselves and how they behave with their binding partners. Indeed, with RIPPLiT, it will be possible to investigate how these interactions and the global mRNP conformation affect the functions of protein complexes, and vice-versa. RIPPLiT may also provide an opportunity to further uncover fundamental differences and similarities between non-coding and protein-coding RNAs, and may be a useful platform to study, potentially using RNA-guided interference experiments, how (m)RNP particles dynamically behave in different cellular contexts.

Figure:

This figure shows the schematic of the RIPPLiT workflow applied to the EJC in a mammalian cell line. Here, the authors adapt their previously developed technique (see Singh et al., Methods 2014, doi: 10.1016/j.ymeth.2013.09.013) for the initial steps of the protocol, followed by ligation of proximal RNA ends and sequencing. – This is Figure 1A from the preprint, made available under a CC-BY-NC-ND 4.0 International license.

Tags: 3d conformation, contact frequency, mrnp organization, ripplit, technique

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