Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes

Abstract

Antibody derivatives, such as antibody fragments (Fab) and single-chain variable fragments (scFv), are now being used to image traditionally hard-to-see protein subpopulations, including nascent polypeptides being translated and post-translationally modified proteins. This has allowed researchers to directly image and quantify for the first time (1) translation initiation and elongation kinetics with single-transcript resolution and (2) the temporal ordering and kinetics of post-translational histone and RNA polymerase II modifications. Here we review these developments and discuss the strengths and weaknesses of live-cell imaging with antibody-based probes. Further development of these probes will increase their versatility and open up new avenues of research for dissecting complex gene regulatory dynamics.

Overlooked Benefits of using Polyclonal Antibodies

“…For instance, epitopes on targets that represent a small change such as a single amino acid polymorphism [34,35] or a post-translational modification [36,37] (e.g.,phosphorylation, methylation, hydroxylation), may be better targeted using a mAb. Often these types of targets or changes are too small to elicit a robust pAb immune response and in addition increase the risk of nonspecific antibody reactivity within the pAb mixture.…”

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

“…2C). The design of this construct allows for detection of the nascent protein with an anti-FLAG antibody fragment (Fab), labeled with green fluorescent Alexa488, as established previously 22,26 . Our reporter gene allows for correct protein translation, confirmed by accumulation of SM-KDM5B labeled with green Fab in cell nuclei ( Supplementary Fig.…”

“…Recently, the MS2 system was combined with antibody-based probes to gain insights in the mRNA life cycle. Translation of single mRNAs can be monitored by following the nascent peptide chain via a loaded affinity probe 22 or the SunTag [23][24][25] ; these studies from different labs yielded comparable protein translation kinetics (see ref 26 for a comparison). Other aspects of the mRNA life cycle, such as interactions with stress granules and P bodies 27 , association with the endoplasmic reticulum 28 , mRNA frame shifting 29 and nonsense-mediated mRNA decay 30 , were also characterized using a combination of MS2-based mRNA labeling and complementary fluorescent probes on the single molecule level.…”

“…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 . While delivery of probes into cells may be technically challenging, advances to improve delivery routes of various fluorescent molecules in live cells have been explored 26,45,46 . Despite these advances, the growing field of RNA biology is in need of diverse and robust fluorescence tools to visualize RNAs with single molecule detection in live cells to gain insights in the complexity and regulation of RNA dynamics.…”

Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

“…One way to resolve post-translational modifications to RNAP2 is to use antibody-based probes that bind and light-up specific modifications to residues within the CTD in vivo [22][23][24][25][26] . How-ever, the signal-to-noise is limited with this approach because of the presence of unbound and freely diffusing probes that increase the fluorescence background.…”