Methods for studying non-coding RNA

Research interest in noncoding RNAs and their biological implications in a variety of cellular contexts has been growing. In this issue, we present a series of pieces discussing recent method advances and future directions for deciphering the regulatory roles of noncoding RNAs.

We highlight the expanding world of noncoding RNA biology in a Collection of articles from Nature research journals that discuss recent technological advances, approaches and emerging models driving this rapidly advancing field.

The research community focused on noncoding RNAs keeps growing. Skepticism about the field has some history.

Researchers roll up their sleeves for the work ahead in the noncoding RNA field.

Recent studies have revealed multifaceted roles of long noncoding RNAs (lncRNAs) in gene regulation, accompanying an increased understanding of lncRNA processing, localization, interacting macromolecules and structural modules. Here, progress and recently developed technological advances for understanding lncRNA biogenesis, modes of action and cellular phenotypes are highlighted, and challenges and opportunities towards higher-resolution and in vivo studies in this field are discussed.

In recent years, the number of annotated noncoding RNAs (ncRNAs) and RNA-binding proteins (RBPs) has increased dramatically. The wide range of RBPs identified highlights the enormous potential for RNA in virtually all aspects of cell biology, from transcriptional regulation to metabolic control. Yet, there is a growing gap between what is possible and what has been demonstrated to be functionally important. Here we highlight recent methodological developments in the study of RNA–protein interactions, discuss the challenges and opportunities for exploring their functional roles, and provide our perspectives on what is needed to bridge the gap in this rapidly expanding field.

Nanopore direct RNA sequencing (DRS) reads continuous native RNA strands. Early adopters have used this technology to document nucleotide modifications and 3′ polyadenosine tails on RNA strands without added chemistry steps. Individual strands ranging in length from 70 to 26,000 nucleotides have been sequenced. In our opinion, broader acceptance of nanopore DRS by molecular biologists and cell biologists will be accelerated by higher basecall accuracy and lower RNA input requirements.

This Review summarizes recent methodological advances in experimental and computational tools developed in studying RNA structures, which provides a bridge for communication between both experimentalists and computational experts.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs with essential roles in germ line development through silencing of transposable elements and in regulation of protein-coding genes. Recent studies have deepened our understanding of the biogenesis and function of piRNAs and their roles in infertility, cancer and neurological diseases in humans.

Le et al. review various technologies for imaging RNAs in fixed and live cells and the biological insights gained from these technologies.

This Perspective focuses on experimental techniques developed for circRNA studies, and provides practical guidelines for performing circRNA research.

Unfried and Ulitsky discuss recent advances in understanding how long noncoding RNAs expressed at much lower levels compared with their targets or cofactors overcome the stoichiometric disadvantages and exert their cellular functions.

Shi et al. discuss recent approaches for the discovery of small noncoding RNAs (sncRNAs), limitations associated with sncRNA expression analyses, and emerging methods for direct and simultaneous detection of multiple RNA modifications.

Mammalian RNA polymerase II transcribes protein-coding genes and non-coding transcription units, including long non-coding RNAs (lncRNAs). Studies applying recently developed nascent transcriptomics technology have revealed differences in transcription initiation and termination between lncRNAs and protein-coding genes, bearing relevance to genomic stress and DNA damage.

Transfer RNAs (tRNAs) are heavily modified post-transcriptionally, and the number and types of modifications are continually expanding. Recent studies show that tRNA modifications can be altered in response to cellular and environmental stresses, and that deficiencies in tRNA modification can cause mitochondrial diseases, neurological disorders and cancer.

Virtually all studies depend on annotations, maps of the genome that catalogue gene loci and the sequences of their transcripts. This Review discusses the state of currently available long non-coding RNA annotations and the impact of emerging technologies such as long-read sequencing.

Loci that encode long non-coding RNAs (lncRNAs) can be complex and function through multiple modalities. The authors provide a framework for elucidating the physiological roles of lncRNAs using genetically engineered mouse models, including whole-gene deletion, transcription termination, reporters and transgene rescue strategies.

This work presents a piRNA-mediated interference for multiplexed gene silencing in Caenorhabditis elegans.

The human transcriptome from 300 tissues and cell lines reveals novel non-coding RNAs.

Xue and colleagues developed LACE-seq to globally profile RNA targets of RNA-binding proteins at single-nucleotide resolution in low-input cells or even single oocytes.

AQRNA-seq allows accurate quantification of small RNAs.

Shi et al. profiled small non-coding RNAs (sncRNAs) through PANDORA-seq, which identified tissue-specific transfer RNA- and ribosomal RNA-derived small RNAs, as well as sncRNAs, with dynamic changes during induced pluripotent stem cell reprogramming.

Gao et al. developed a CRISPR–Cas9-based system in which sgRNA production is controlled by the endogenous promoter to monitor the expression of weakly expressed genes and long non-coding RNAs in mammalian cells.

SEEKR is a method that deconstructs linear sequence relationships between lncRNAs and evaluates similarity on the basis of abundance of short motifs called k-mers. LncRNAs of related function often have similar k-mer profiles despite lacking linear homology.

This paper describes a CRISPR–Cas13 system to effectively target circRNAs and screen their functions in vitro and in vivo, which enables the study of relevant circRNA phenotypes in human cell proliferation and in mouse embryogenesis.

Femtosecond XFEL crystallography is used to identify dynamic changes in the adenine riboswitch aptamer domain, with at least four states identified in real time, two in the apo form before binding and two with the ligand bound.

ARM-seq enables enhanced sequencing of modified tRNAs and tRNA fragments. Treatment of RNA with the demethylase AlkB prior to reverse transcription removes three ‘hard-stop’ modifications, allowing for discovery of modified tRNA fragments and their precursors by RNA sequencing.

The combination of an engineered demthylase and a highly processive reverse transcriptase during tRNA library preparation for high-throughput sequencing allows comprehensive profiling of the small RNAs.

A catalogue of human long non-coding RNA genes and their expression profiles across samples from major human primary cell types, tissues and cell lines.

A natural circular RNA termed ciRS-7 is shown to function as a negative regulator of microRNA; ciRS-7 acts as an efficient sponge for the microRNA miR-7, and is resistant to the usual microRNA-mediated degradation pathway of exonucleolytic RNA decay.

Biochemical, functional and computational analyses are combined to show that circular RNAs are a large class of animal RNAs with regulatory potency.

A description is given of the ENCODE effort to provide a complete catalogue of primary and processed RNAs found either in specific subcellular compartments or throughout the cell, revealing that three-quarters of the human genome can be transcribed, and providing a wealth of information on the range and levels of expression, localization, processing fates and modifications of known and previously unannotated RNAs.

This study uses chromatin marks in four mouse cell types to identify ∼1,600 large multi-exonic transcriptional units that do not overlap known protein-coding loci and are highly conserved. Putative functions are assigned to each of these large intervening non-coding RNAs, which range from ES pluripotency to cell proliferation.