Regulatory Non-Coding RNAs: Methods and Protocols

Computational Biology of Non-Coding RNA
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We have a dedicated site for Germany. Regulatory Non-Coding RNAs: Methods and Protocols offers a collection of methods for those interested in the discovery, localization, and functional analysis of these non-coding transcripts that have the potential and ability to orchestrate and control gene expression. After a review of the field, this detailed volume continues with methods useful for the study of siRNAs, microRNAs and their targets, techniques concerned with long non-coding RNAs, as well as studies of the critical parameters of functional non-coding RNA protein-RNA interactions and the environment in which they act.

Written for the highly successful Methods in Molecular Biology series, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips for troubleshooting and avoiding known pitfalls. Dependable and easy to use, Regulatory Non-Coding RNAs: Methods and Protocols provides a current, state-of-the-art collection of methods and approaches that will be of value to researchers in this expanding and fascinating field.

JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser. This article reviews fundamental insights into the inter-regulation of enhancers and promoters and discusses unresolved questions regarding the functional role of enhancers as transcription units in genome regulation. This Review discusses how, beyond their lack of protein-coding potential, some types of lncRNAs are known to exhibit features that are distinct from mRNAs, including their transcriptional regulation, localization, processing, biological capabilities and degradation.

Such properties underlie many of the key cellular functions of lncRNAs. This Review describes how molecular studies and structural data are revealing key insights into both mechanisms of miRNA-mediated gene repression in animals, including their intricate interplay, and are posing new questions for future research. This Review describes our latest understanding of the range of functions of tRNAs. Emerging roles include the tight regulation of tRNA biogenesis to meet the translational needs of different cell types, cleavage and covalent modification of tRNAs in stress signalling, and diverse mechanistic links to various diseases.

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Small and long non-coding RNAs have emerged as key regulators of gene expression through their direct and indirect actions on chromatin. This Review describes how RNAs form powerful surveillance systems that detect and silence inappropriate transcription events, and how such systems provide a memory of these events via self-reinforcing epigenetic loops. This Review highlights the challenges and solutions of this point of view, particularly for the synthesis and replication of RNA, and how various types of molecular cooperation probably had important roles.

It also highlights the role of computational modelling in furthering the conceptual understanding of miRNA functions in gene regulatory networks. Discoveries over the past decade portend a paradigm shift in molecular biology; evidence suggests that RNA is not only functional as a messenger between DNA and protein but also involved in the regulation of genome organization and gene expression. This Timeline article surveys the emergence of the previously unsuspected world of regulatory RNA from a historical perspective.

Timeline 29 Apr Nature Reviews Genetics. This Review discusses our latest understanding of lncRNAs that have validated functional roles in various differentiation and developmental processes. Antisense transcription is increasingly being recognized as an important regulator of gene expression across all kingdoms of life and through a range of regulatory modes. Antisense transcripts are also emerging as facilitators of rapid evolution of gene regulation. This Review discusses roles for microRNAs in adult animals — including in adult stem cells, metabolism and in cancer — and how functions in adults can be distinguished from developmental roles using a range of methods.

Understanding adult-specific functions has implications for therapeutic manipulation of miRNAs. This Review describes our latest understanding of various steps in this process, from the specification of piRNA-producing loci to piRNA processing and nuclear effector functions, including a role in transgenerational epigenetic inheritance. Argonaute proteins are vital components of small-RNA-guided modes of gene regulation.

Recent studies have provided important details about classical modes of Argonaute function, such as their structure and loading with small RNAs, and have also revealed unexpected roles in other cellular functions.

Regulatory Non-Coding RNAs

In addition to well-known roles in the cytoplasm, a growing number of functions for small RNAs in the nucleus are being discovered. These include roles in transcriptional repression, epigenetic modifications and genome stability. This Review considers examples from animals, plants and fungi. Owing to the important role of microRNAs in gene regulation, profiling repertoires of expressed microRNAs can be informative in basic research and clinical settings. Following the identification of lncRNAs, it is imperative to determine whether these non-coding transcripts indeed possess biological functions or not.

Nuclear Bodies and Noncoding RNAs—Methods and Protocols

Annu Rev Genet. Humoral immunity is a multilayered process that involves activation and maturation of B cells. Top 10 differentially expressed lncRNAs in psoriasis tissues compared with normal control. Unique features of long non-coding RNA biogenesis and function. Jomar F.

To ascertain the physiological roles of lncRNAs in the cell, experimental studies with perturbation of lncRNA expression are necessary in order to reveal the contribution of an lncRNA to particular phenotypes e. Although gain-of-function studies may reveal the significance of trans-acting regulatory roles for lncRNAs, loss-of-function approaches still represent the standard and most common strategy to investigate the function of a gene in reverse genetics [ 43 ]. With extensive prior experience of mRNA knockdown and great advancement of genome manipulation technologies, there are already a variety of choices of knockdown or knockout methods available for lncRNAs.

In this review, the most common genetic manipulation approaches will be discussed in the next section and their conceivable readouts related to hematological disorders will be discussed in the section Functional targets for probing biological effects of lncRNAs in blood cancer cells. RNAi approaches generally make use of transcripts 20—40 nt in length and complementary to the target RNA transcript.

Upon binding, the subsequently formed duplexes will then be degraded via cellular machinery [ 44 ]. These approaches have been extensively applied mainly because they are relatively fast and easy to use.


In essence, they are cost-effective and can be specifically engineered to target an RNA sequence in a precise manner. One strategy is to transfect target cells with small interfering RNAs siRNAs that target the transcript of interest in a transient fashion while another is to introduce short hairpin RNAs shRNAs that are stably expressed. On the contrary, the shRNA strategy provides sustained knockdown of the lncRNA target, and hence is more suitable for experiments investigating the prolonged effects of targeted lncRNA depletion [ 45 ].

Although RNAi approaches are widely used with many successful examples, concerns have been raised about the effectiveness of these strategies for the depletion of nuclear and enhancer-associated lncRNAs. It has been argued that these lncRNAs predominantly localize in the nucleus and this impedes their susceptibility to the RNAi machinery, which is primarily located in the cytoplasm [ 46 ]. Consequently, several siRNA sequences are usually screened to find out a more potent one for the effective knockdown of a specific lncRNA. ASOs are able to efficiently degrade nuclear lncRNAs via a mechanism dependent on ribonuclease H, leading to depletion of nascent transcripts.

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However, these oligo-based methods still share some drawbacks with RNAi, including incomplete knockdown, unpredictable off-target effects, and transient inhibition effects. All these impose limitations on loss-of-function analysis of lncRNAs.


Nevertheless, given the relatively straightforward manner of operation, RNAi- and ASO-mediated knockdown strategies remain a helpful and valuable tool for the initial investigation of lncRNA functionality. To address the limitations of RNAi and ASOs, programmable nuclease-directed genome-editing methods provide a powerful alternative approach to characterizing the functional roles of lncRNAs both in vitro and in vivo [ 50 , 51 ]. This genome-editing tool is now able to be carried out at the DNA level and in an efficient and rapid manner to achieve partial or total deletion of lncRNA loci Fig.

In short, the system makes good use of an endonuclease called Cas9, which is directed by a specially designed single-guide RNA sgRNA to the desired site and then performs a site-specific cutting at a target gene i. A popular way to knock out a protein-coding gene by using CRISPR-Cas9 is via frameshift mutations that can be easily induced at an ORF by Cas9-mediated cleavage followed by a non-homologous end-joining repair.

However, it may not be applicable to the generation of a knockout effect for non-protein-coding genes because of their non-coding nature as well as our limited knowledge about their functional mechanisms.

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In general, most sequences particularly responsible for the molecular functions of lncRNA transcripts have not yet been characterized. In addition, some lncRNAs exert their functions by the act of transcription per se instead of the lncRNA transcript [ 54 , 55 ]. In that case, genetic manipulation should specifically target regulatory regions controlling the transcription, which are often poorly annotated or remain largely unknown, leading to more challenges when studying such type of lncRNAs.

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Therefore, more comprehensive approaches tailored to lncRNA are necessary and will be discussed below. The first common strategy utilizes two distinct guide RNAs to simultaneously target two specific locations flanking the lncRNA gene of interest, and hence removes the entire genomic locus encoding that lncRNA by Cas9-mediated cleavage Fig. This ensures complete and permanent ablation of the lncRNA by deleting the whole genomic region associated with it.

Besides, this approach has already been extended to genome-wide scale for high-throughput lncRNA depletion screening in human cancer cells [ 56 ]. Another alternative strategy is to solely delete the core promoter region of the given lncRNA gene in order to abolish its transcription Fig. This approach has two major merits over the conventional removal of a whole gene. First, it has been shown that there is an inverse relationship between the size of a target region being cut and the efficiency of excision [ 58 ].

By removing the promoter region alone, the deletion size is in the range of 0. It has also been shown that promoter excision could reduce lncRNA expression more effectively than the removal of an individual exon, intron, or splice site [ 59 ]. Second, observed phenotypic changes can be more confidently ascribed to the absence of that particular lncRNA, but not an unintended result of removing any overlapping gene or regulatory elements around the targeted genomic region, which is a major concern of the approach based on whole gene deletion [ 52 ].

Nevertheless, a study reported that inhibiting lncRNA MALAT1 expression was solely achieved by deleting its major promotor, but not the annotated upstream promoter [ 53 ]. This implies that if there are multiple known promoters for a given lncRNA, it would be better to test all of them to come up with one having the best performance.

Nature Reviews Genetics

However, unlike the case of coding genes, lncRNA promoters are often poorly annotated and this may hamper the experimental design when using such a strategy. As the current annotation status of most ncRNAs is still provisional, it is difficult to determine the essential promoter or exon regions to be knocked out. Therefore, a newly tested approach is to achieve knockout through the excision of a TSS signature under the guidance of epigenetic data [ 60 ]. The chromatin condensation state of eukaryotic genes can influence the binding or interaction of polymerases and transcription factors and such epigenetic alteration is actively regulated through histone methylation, acetylation, or phosphorylation.

For instance, histone H3 lysine 4 trimethylation H3K4me3 localized near TSS typically signifies regions of active transcription while H3K4me1 and H3K27ac are the predominant histone modifications around active enhancer elements. From the data of high-throughput epigenetic profiling, gene-proximal H3K4me3 modification sites frequently coincide with DNaseI hypersensitivity sites DHSs , which indicate accessible chromatin regions [ 61 ]. As a general caution, excision of a genomic DNA sequence may have unexpected impact on the expression of neighboring genes.

Bidirectional transcription is an example that transcription from both DNA strands can be suppressed by deleting the shared promoter segment, which may confound the interpretation of an observed phenotype. In addition to knockout approaches by excision of genomic regions, nuclease cleavage technology can be further utilized to achieve lncRNA knockdown by knocking in destabilizing elements or transcriptional stop signal into the gene i.

Table of contents

Regulatory Non-Coding RNAs: Methods and Protocols offers a collection of methods for those interested in the discovery, localization, and functional analysis of. Long Non-Coding RNAs: Methods and Protocols also discusses methods used to . Long noncoding RNAs (lncRNAs) are a new class of regulatory genes that.

A recent study reported successful silencing of an lncRNA gene by biallelic insertion of a poly A signal into the genomic locus via CRISPR-Cas9-mediated double-strand break followed by a homology-directed repair [ 63 ]. In the study, three different insertion targets for poly A integration were suggested, including the first intron, inside the first exon, and the region immediately after the promoter.

This provided different choices of integration sites to achieve the best suppressing effect for a particular target gene. Besides, a closer look into the results revealed that gene silencing through the introduction of a poly A signal did not entirely abolish the transcription of the target lncRNA gene. This feature would be helpful for functional studies of genes that may cause lethal phenotype upon complete knockout.

Another study wisely harnessed the flexibility of CRISPR-Cas9 system to apply to genomic locus such modifications as deletion or insertion of stop signal, strong constitutive promoter, and even cDNA rescue in experiments. The integrated analysis revealed opposing roles for the transcript of lncRNA Haunt and its genomic locus in the regulation of HOXA gene clusters [ 55 ]. Furthermore, compared to knockout approach, knocking in the termination signal together with selectable marker e.

RNA fluorescence in situ hybridization in cultured mammalian cells

Such development repurposed the utility of Cas9 protein to become an amenable DNA-targeting modular scaffold. In the simplest way, dCas9 can be used to block the binding of transcription factor or RNA polymerase by steric hindrance; this in turn hampers transcriptional initiation and elongation, and results in a knockdown effect [ 65 ]. Meanwhile, such epigenetic silencing of gene expression, like genomic region deletion, acts at the DNA level so that it could target lncRNAs without competing with endogenous RNA machinery when compared to RNAi methods.

After all considerations, the major limitation of using CRISPR-Cas9 to target lncRNAs is the complex architecture of genomic loci surrounding different lncRNA genes, which may highly increase the possibility of disturbing neighboring or overlapping genes. This may greatly reduce the specificity of genetic manipulation of lncRNAs and lead to a false positive phenotypic change resulting from the intervention of neighboring genes.

With consideration of such constraint, the targeted lncRNA loci should be carefully studied before the design of sgRNAs, and the expression of neighboring genes should also be monitored in parallel. Consequently, different experimental techniques should be taken into account to complement each other and this may greatly facilitate the study of lncRNA functions. Although there is still no application example to target lncRNA, it is anticipated that the Cas13 system will be utilized for lncRNA knockdown study owing to their high specificity and flexible utility to be mentioned below Fig.

In an assessment of various Cas13 variants, LwaCas13a from Leptotrichia wadei was identified as the most active Cas13a ortholog for targeted RNA knockdown in human cells. In general, some Cas13a orthologs need a protospacer flanking site PFS analogous to the protospacer adjacent motif site for Cas9 system, but there was no such constraint for LwaCas13a.