Functional RNA Glyphs

[jblucks]

Current SBOL RNA-related glyphs (i) do not capture the range of RNA functions and (ii) where they do exist are not expressive enough to precisely annotate the RNA function. A new set of glyphs that more precisely expresses RNA functions are needed to capture the repertoire of available functional RNA systems.

For example, an increasingly used tool in synthetic biology are riboswitches - cis-acting RNA element that binds to a specific chemical target via an aptamer domain, then undergoes a structural transition to control a specific aspect of gene expression (transcription, translation, splicing) via an expression platform domain). When wanting to annotate a riboswitch, the aptamer glyph is not precise enough as it does not specify the type of gene expression process regulated by the aptamer. This is important as a riboswitch that controls transcription has different implications for genetic circuit function than one that controls translation. One possible solution
is to create a fusion between the aptamer glyph and the terminator glyph to indicate that the riboswitch controls transcription, or a fusion between the aptamer glyph and the RBS glyph to indicate that the riboswitch controls translation. It could be extended too - for example if a splice site glyph was determined then the aptamer glyph and this glyph combined could be used for a riboswitch that controls splicing.

A system of RNA glyphs is needed to capture riboswitches and other increasingly used functional RNA elements such as:

• trans-acting RNAs including but not limited to: toe hold switches, small RNA regulators of translation, small transcription activating RNAs
• cis-acting RNAs including but not limited to: riboswitches, splice sites and splicing regulators
• RNAs that act via protein binding mechanisms

Ideally this glyph system could be easily adapted as new types of functional RNAs are created.

[JS3xton]

A great proposal, and one that has been called for previously (here, here, here, here, and here).

In the past, a good starting point has been to collect examples (images) from the literature to guide glyph development. Talented artists can then begin to iterate on glyph designs. As glyphs mature, they can be cataloged in one (or more) SBOL Enhancement Proposal(s) (SEPs) and then voted on.

Many functional RNA elements are listed, which may be difficult to manage in one thread. Separate GitHub issues may help to organize the development of different functional RNA elements.

I would also like to incorporate a few other improvements from another thread that I believe are relevant. In particular, @shyambhakta noted that the Aptamer glyph does not show the aptamer binding anything, as it should. I also proposed a new glyph for self-cleaving ribozymes.

To begin, I recommend folks familiar with these technologies first agree on different classes of functional RNA elements (perhaps using those laid out by @jblucks above), and then present literature examples, from which glyph artists can begin to distill common SBOL Visual-compliant representations.

[lcg5]

As Julius mentioned above, we think combining current glyphs would potentially be the best way to describe many RNA technologies. Below, we have compiled a non-exhaustive list of current RNA technologies. These systems are split into three types of regulation: Transcriptional, Translational, and Cleavage-based control. The “Components” column describes what RNA motifs the regulators are composed of. This shows that many components overlap across the different types of regulation. Riboswitches, for example, all contain an aptamer and a second component that describes the type of regulation it controls. As a result, we envision combined glyphs to be the most modular, yet detailed way to describe these important differences. Shown below, transcriptional riboswitches can be described by a combined aptamer and terminator glyph, translational riboswitches by combined aptamer and RBS glyphs, and cleavage-based riboswitches by combined ribozyme and aptamer glyphs (as discussed earlier by John Sexton).

As for other RNA regulatory systems, new glyphs might be required. For the Small Transcription Activating RNA (STAR) technology, a small RNA binds to a transcriptional terminator located upstream of a gene. This binding event disrupts the terminator and allows for transcription to continue, enabling activation of the gene of interest. The terminator glyph would effectively describe this genetic construct, but it doesn’t capture the switchable nature of this technology. In addition, though the ligand-binding event can be adequately described by interaction glyphs (i.e. activation/repression), this technology would benefit from showing that the terminator can be bound by a small RNA.

With this in mind, we would like to propose a new ncRNA binding site glyph. This glyph can be combined with a terminator (above) to describe transcriptional regulators like STARs, attenuators, and CRISPR regulators. It can also be combined with the RBS glyph to describe translational technologies like Toehold switches and antisense RNAs.

We welcome feedback from the SBOL community on this proposal.

Thanks
Lauren Gambill
James Chappell

Type	Regulator	Components	Modality of regulation	Example Citations
Transcriptional	Riboswitch	Terminator, aptamer	Activation/Repression	Ceres (2013), Wachsmuth (2013)
Transcriptional	STAR	Terminator, ncRNA, ncRNA binding site	Activation	Chappell (2015, 2017)
Transcriptional	Attenuator	Terminator, ncRNA, ncRNA binding site	Repression	Lucks (2011), Takahashi (2013)
Transcriptional	CRISPRa/i	dCas9, sgRNA, sgRNA binding site	Activation/Repression	Qi (2013), Bikard (2013), Dong (2018)
Translational	Riboswitch	RBS, aptamer	Activation/Repression	Espah (2016)
Translational	Toehold switch	RBS, ncRNA, ncRNA binding site	Activation/Repression	Green (2014), Kim (2019)
Translational	Antisense RNA	RBS, ncRNA, ncRNA binding site	Repression	Mutalik (2012), Isaacs (2004),Rodrigo (2012)
Cleavage	Cis-cleaving riboswitch	Cis-cleaving ribozyme, aptamer	Stability, Activation/Repression	Lou (2012), Felleti (2016)
Cleavage	Cis-splicing riboswitch	Cis-splicing ribozyme, ligand, exon, intron	Activation/Repression	Winkler (2004), Lee (2010)
Cleavage	Trans-splicing riboswitch	Trans-splicing ribozyme, ligand	Activation/Repression	Köhler (1999), Müller (2017)
Cleavage	Protein cleavage	Endonuclease, binding site	Repression	Przybilski (2011)
Cleavage	CRISPR	sgRNA, Cas9	DNA insertions/deletions	Pyne (2015)

[shyambhakta]

I stumbled across a figure that perfectly inspires a modular composite RNA part glyph. @lcg5
From Win & Smolke (2008)

[jakebeal]

Hi, folks: I am revisiting this after a while and have been pondering two families of questions with respect to these diagrams.

1. How geometric are the diagrams?
   • I see a lot of right angles, straight lines, and 45 degree angles. If one bent them, would the meaning change?
   • I see a lot of symmetry in diagrams. Does this always indicate literal symmetry in the expected structure?
2. How much are the diagrams to scale?
   • If I see 3 strokes in a double-stranded region, does that literally mean 3 bases?
   • If I see a bubble, can I compute the number of bases from the length of the bubble edges?