Single molecule fluorescence approaches shed light on intracellular RNAs.

The eukaryotic cell is highly complex. Ever since Robert Hooke discovered “cells” in 1665 when training his comparably primitive microscope on a sliver of cork, scientists have aimed to identify and characterize all functional components of the cell. Around the turn of the millennium, the Human Genome Project laid open our entire cellular catalogue, but shockingly discovered that less than 21,000 protein-coding genes – just ~5-times the number of a bacterium such as Escherichia coli – span only ~1.2% of the over 3 billion base pairs of the human genome.1-4 This lack of proteomic inventory initially perplexed the scientific community, but then spurred debates of possible underlying RNA contributions to cellular complexity.5,6 The Encyclopedia Of DNA Elements (ENCODE) project, an international collaborative research effort, was initiated to provide a comprehensive picture of all functional elements within the human genome through unbiased, transcriptome-wide coverage by RNA deep-sequencing (RNA-seq).7 Particularly striking are the discoveries that at least 75% of the genome is transcribed and that by far most of these transcripts do not code for proteins, but rather “non-coding” RNAs (ncRNAs), many of which are still uncharacterized in terms of their structure and function.7,8 Currently, more than 80,000 distinct ncRNAs have been identified in human cells, which reveals an unexpected and exciting RNA landscape in our body (with excerpts highlighted in Figure 1).9 Many RNA elements have been found to originate from overlapping loci, suggesting that similar RNA sequences can be distinctly generated or processed to perform different biological functions.10,11 In an effort to understand the complex functional networks these RNAs are involved in, systems biology approaches are beginning to be implemented. Abetting such holistic approaches are single molecule methods that promise to provide quantitative mechanistic details for individual biomolecules within living cells. Figure 1 Survey of the RNA biology in a eukaryotic cell While RNA-seq has proven powerful for discovering novel cellular RNAs, the approach is limited by the ensemble averaging and loss of spatiotemporal information caused by the isolation of cellular RNA. It thus remains unclear whether, for example, functionally important ncRNAs are expressed in low quantities across all cells of a sample or selectively expressed only in a few cells, which feigns low expression by dilution within the averaged measurement. Single molecule approaches have emerged as an unparalleled means to resolve complex cellular processes that are otherwise masked by such ensemble averaging. The recent implementation of single molecule fluorescence tools to characterize of mRNA expression rates and levels, mRNA and microRNA localization, and ribonucleoprotein complex (RNP) association in living cells, together with the emergence of super-resolution imaging techniques such as PALM and STORM,12 endows single molecule techniques with the potential to broadly dissect the functions and mechanisms of ncRNAs. In this review, we begin with an overview of the different classes of RNAs in eukaryotic cells, in terms of their biogenesis, function and localization (Figure 1). Given the extraordinary amount of literature on these subjects, where appropriate we guide the reader to pertinent reviews for further detail. Next, we summarize recent technical achievements of single molecule fluorescence microscopy in visualizing RNA and RNA-protein complexes in vivo. Finally, we highlight some applications of single molecule tools over the last 15 years that investigate RNA function within cells. Throughout the text, we will promote a vision of uniquely resolving the still shrouded multitude of functional mechanisms of RNAs, especially ncRNAs, through single molecule approaches.