In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

Abstract

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…They note that triphosphates bearing modificationsa tt he C2',C 3 ', and C8 positions are not accepteda ss ubstrates by the TdT. On the other hand, upon using the yeast poly(A)p olymerase (PAP) [125] instead, efficient tailing reactions are observed with most rN*TPs. [126] These two examples showt hat engineered versions of the TdT (or relatedp olymerases) might be necessary for reliable and robust tailing reactions of RNA oligonucleotides.…”

Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

“…Such alterations to thermal stability can potentially have functional consequences. In the context of miRNA guide strand selection and loading into the RNA-induced silencing complex (RISC), for example, a 5 ′ terminal fluorophore disfavored use of the labeled strand as a guide (Custer and Walter 2017). Further, in contrast to DNA where canonical Watson-Crick base-pairing dominates, RNA secondary and tertiary structures show an astonishing variety of noncanonical base-pairing interactions (Leontis et al 2002), including interactions with the sugar edge (Nissen et al 2001).…”

“…2B and D). For eukaryotic mRNAs, fluorophores can be incorporated into mRNAs in the cap structure (Mamot et al 2017), randomly incorporated throughout the transcript (Schulz and Rentmeister 2014), in the poly(A) tail, or between the 3 ′ UTR and poly(A) tail (Custer and Walter 2017). Such covalent attachment of the fluorophore allows labeled molecules to be detected with greater sensitivity, without the background of free fluorophores that is a recurrent challenge of RBP-based RNA detection.…”

“…Such covalent attachment of the fluorophore allows labeled molecules to be detected with greater sensitivity, without the background of free fluorophores that is a recurrent challenge of RBP-based RNA detection. As with RBP-mediated labeling, however, there can be functional consequences to these covalent labeling strategies; for example, random fluorophore incorporation throughout the transcript was shown to dramatically impair the coding function, and incorporation of fluorophores in the 3 ′ UTR was shown to stabilize mRNAs (Custer and Walter 2017).…”

Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

“…More recently developed techniques to label RNA use smaller aptamer tags that bind small molecules, including the Spinach/Broccoli 34,35 and Mango tags 36,37 , but single molecule detection in living cells has not yet been achieved with any small molecule-based approach. Entirely orthogonal tools have been developed in which fluorescent probes are delivered into cells to bind untagged RNA without the need to genetically add tags [38][39][40] , enabling in principle visualization of any endogenous RNA of any size, such as miRNA 41,42 . Finally, additional approaches for tracking RNAs include incorporation of fluorescent nucleotide analogs to study mRNAs encoding mitochondrial proteins translated on endosome-containing 'hotspots' in axons 43 , molecular beacons for studying co-transcriptional vs. post-transcriptional alternative splicing 44 , and micro-injected fluorescent RNA probes to examine the miRNA life cycle 41,42 .…”