Noncoding RNA

David P. Clark , ... Michelle R. McGehee , in Molecular Biology (Third Edition), 2019

Abstract

Noncoding RNA refers to all those RNA molecules that play biological roles as RNA, rather than being translated into protein like messenger RNA. Transfer RNA and ribosomal RNA take part in protein synthesis. Occasional RNA molecules act catalytically, as RNA enzymes (ribozymes) or take part in protein export. Two other major roles for noncoding RNA are in genome defense and in genetic regulation. Small noncoding RNA molecules take part in RNA interference in eukaryotes and in CRISPR in prokaryotes. Both systems protect against viruses and other hostile genetic elements. A wide variety of noncoding RNA molecules, both long and short, are involved in regulating gene expression. There are major differences between prokaryotes and eukaryotes. Prokaryotes use relatively few regulatory RNAs and these are usually short. Eukaryotes have a much greater variety of regulatory RNA molecules, including long noncoding nuclear RNA (lncRNA). In addition, in eukaryotes, several classes of small RNA take part in the processing of mRNA, tRNA and rRNA inside the nucleus.

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Nanostructured Materials for DNA Biochip

Bansi Dhar Malhotra , Md. Azahar Ali , in Nanomaterials for Biosensors, 2018

8.2.2 RNA

RNA consists of an unbranched single-strand polymer with many intramolecular double-stranded sections. The backbone of RNA is formed by 3′, 5′-phosphodiester bonds, the sugar is ribose, and the nucleobase is thymine. Owing to steric hindrance caused by the 2'-OH groups of ribose, the double-stranded RNA cannot be formed. Using an RNA genome, many viruses are known to encode their genetic information. Four functional RNA families are known as ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA, and small nuclear RNA (only present in eukaryotes). RNA is known to play various roles such as in coding, decoding, regulation, and expression of genes. Fig. 8.1.7 shows the structural difference of RNA with DNA.

Figure 8.1.7. Schematic representation of the structure of RNA (left-hand side) and DNA (right-hand side) with its nitrogenous bases.

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Analysis of Gene Expression

David P. Clark , Nanette J. Pazdernik , in Molecular Biology (Second Edition), 2013

6.1 Assessing the Purity of RNA

RNA purity is essential for all the different transcriptomic procedures. Most RNA samples are obtained from the cells of interest by either isolating total RNA or mRNA with a poly(A) tail. In either case, rRNA accounts for the majority of all the RNA in a cell. Its abundance can mask the other types of RNA, and therefore, rRNA must be removed. One efficient method to remove the ribosomal RNA is to hybridize an rRNA probe labeled with a biotin tag to the sample of total RNA. The hybrids are then removed from the remaining RNA by binding to streptavidin-coated magnetic beads. The remaining RNA is enriched for mRNA and provides a better sample for transcriptome analysis (Fig. 19.18).

Figure 19.18. Removing Unwanted rRNA from an RNA Sample

Although most rRNAs are not polyadenylated, a fraction of the transcripts do have poly(A) tails. These can contaminate RNA for transcriptome analysis, and therefore, need to be removed. One method uses biotinylated single-stranded probes that have complementary sequences to rRNA. These hybridize to the rRNA in the sample and are removed by binding to avidin-coated beads followed by centrifugation.

To ensure that the RNA sample is free of degradation and contamination, a small amount of the sample may be analyzed using lab-on-a-chip methods (see Ch. 8). For example, the RNA 6000 Pico LabChip from Agilent Technologies can analyze nanogram quantities of RNA by electrophoresis through a gel/dye matrix in a small chip (Fig. 19.19). Just as in traditional electrophoresis, the RNA fragments move based on size. The bands are visualized with a fluorescence detector and graphed via an attached computer. The graph can determine if the sample is contaminated with rRNA, and based on the sizes of the peaks, whether or not the sample is degraded.

Figure 19.19. Pico LabChips Separate Nanogram Quantities of DNA or RNA

A) The lab on a chip has small holes in a glass piece that hold the DNA or RNA sample, the size comparison ladder, and the gel materials. The holes are connected via microfluidic channels. B) The scientist adds the experimental sample of RNA or DNA into the specific hole. C) The analyzer then performs the analysis and records the data on an attached computer (not shown).

 (Credit: Reproduced with Permission of Agilent Technologies.)

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RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways

Sarah E. Cole , Frederick J. LaRiviere , in Methods in Enzymology, 2008

Abstract

Mature rRNA are normally extremely stable in rapidly growing cells. However, studies show that some mature rRNA in Saccharomyces cerevisiae are, in fact, turned over quite rapidly by the nonfunctional rRNA decay (NRD) pathway. NRD eliminates the RNA component of mature but defective ribosomal subunits and ribosomes. NRD was discovered using rDNA reporter plasmids to express and track the fate of rRNA containing mutations in functionally important regions of the ribosome. This chapter outlines some of the available rDNA reporter plasmids that can be used to study NRD and describes assays to test for functionality and stability of rRNA in yeast.

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Analysis of Gene Expression

David P. Clark , ... Michelle R. McGehee , in Molecular Biology (Third Edition), 2019

6.1 Assessing the Purity of RNA

RNA purity is essential for all the different transcriptomic procedures. Most RNA samples are obtained from one or more cells of interest by isolating RNA. Depending on the goal of the experiment, different types of RNA can be isolated accordingly. For example, mature mRNA with a poly(A) tail represent coding sequences and can be isolated by binding to poly(T) linkers attached to magnetic beads. This is particularly effective for isolating mRNA from eukaryotic cells since addition of the poly(A) tail is part of RNA processing prior to translation; but requires artificial polyadenylation or creation of linker or primers for prokaryotic transcriptome analysis. Additionally, this method selects against the enrichment of most noncoding RNA because only RNA that has been processed by adding a 3′ tail will be isolated. In practice only mRNA and a few special lncRNAs receive the poly(A) tail. In another example, the binding of complementary oligomer probes linked to a purification tag can isolate targeted transcripts. Even small, noncoding RNAs can be specifically captured using specific 5′ or 3′ end modifications. In any case, rRNA accounts for the majority of all the RNA in a cell. Its abundance can mask the other types of RNA, and therefore, rRNA must be removed. One efficient method to remove the rRNA is to hybridize an rRNA probe labeled with a biotin tag to the sample of total RNA. The hybrids are then removed from the remaining RNA by binding to streptavidin -coated magnetic beads. The remaining RNA is enriched for the desired RNA type and provides a better sample for transcriptome analysis (Fig. 21.17).

Figure 21.17. Removing Unwanted rRNA From an RNA Sample

Although most rRNAs are not polyadenylated, a fraction of the transcripts do have poly(A) tails. These can contaminate RNA for transcriptome analysis, and therefore, need to be removed. One method uses biotinylated single-stranded probes that have complementary sequences to rRNA. These hybridize to the rRNA in the sample and are removed by binding to avidin-coated beads followed by centrifugation.

Different types of RNA can be enriched in RNA samples.

To ensure that the RNA sample is free of degradation and contamination, a small amount of the sample may be analyzed using lab-on-a-chip methods (see Chapter 8: DNA Sequencing). For example, the RNA 6000 Pico LabChip from Agilent Technologies can analyze picogram quantities of RNA by electrophoresis through a gel/dye matrix in a small chip (Fig. 21.18). Just as in traditional electrophoresis, the RNA fragments move based on size. The bands are visualized with a fluorescence detector and graphed via an attached computer. The graph can determine if the sample is contaminated with rRNA, and based on the sizes of the peaks, whether or not the sample is degraded.

Figure 21.18. The Bioanalyzer RNA Pico 6000 assay Separates Picogram Quantities of RNA

(A) The Pico Chip has small holes in a glass piece that hold the RNA sample, the size comparison ladder, and the gel materials. The holes are connected via microfluidic channels. (B) The scientist adds the experimental sample of RNA into the specific hole. (C) The analyzer then performs the analysis and records the data on an attached computer (not shown).

 Credit: © Agilent Technologies, Inc. Reproduced with Permission, Courtesy of Agilent Technologies, Inc.

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RNA-Based Technologies

David P. Clark , Nanette J. Pazdernik , in Biotechnology (Second Edition), 2016

Abstract

RNA plays a multifaceted role in biology that is adaptable for many different applications in biotechnology. The most widely understood role of RNA is in protein synthesis, which includes messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) (see Chapter 2). However, RNA plays many other roles. Several small RNAs, such as snRNA, snoRNA, and gRNA, take part in RNA processing by removing introns. Some RNA sequences can catalyze enzyme reactions. Ribozymes, as they are called, are found in many organisms, catalyzing cleavage and ligation of various substrates. Between the increased speed and accuracy of sequencing and a heightened awareness of RNA in the cell, an ever-increasing number of roles has been found for RNA in the regulation of gene expression and in cell defense. Entirely new classes of noncoding RNAs (ncRNAs) have been discovered and characterized.

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Processing of RNA

David P. Clark , Nanette J. Pazdernik , in Molecular Biology (Second Edition), 2013

1 RNA Is Processed in Several Ways

RNA is made by RNA polymerase, using a DNA template, in the process known as transcription (see Ch. 11). Sometimes the RNA molecule is ready to function immediately after it has been transcribed (e.g., most bacterial mRNAs). However, in many cases, the RNA needs further processing before it is functional. In these cases, the original RNA molecule, before any further processing occurs, is known as the primary transcript. For specific classes of RNA, the precursor (i.e., primary transcript) may be referred to as pre-mRNA, pre-tRNA, etc. The term hnRNA (heterogeneous nuclear RNA) was also used previously, before the relationship of precursor RNA to the final processed RNA product was understood.

Many RNA molecules are modified in a variety of ways after being synthesized.

All classes of RNA are subject to processing by base modification and cleavage. In addition, eukaryotic mRNA undergoes capping and tailing as well as splicing (Fig. 12.01). Base modifications occur primarily in tRNA and rRNA, and occur after the RNA is transcribed. These modifications are essential for their proper function in protein translation (see Ch. 13). Certain RNA molecules such as prokaryotic and eukaryotic rRNA are modified by cleavage; that is, the RNA is made as a longer precursor that is trimmed to the correct length. In other, related cases, several RNA molecules are included in the same primary transcript, which is then cleaved into several parts. In eukaryotes, the primary transcript for mRNA contains segments called introns or intervening sequences that are not used to encode the final protein product (see Ch. 4). Splicing involves the removal of these introns and rejoining of the ends to create a streamlined mRNA with an uninterrupted coding sequence that is translated into a protein.

Figure 12.01. Types of RNA Processing

RNA processing can be divided into cutting/joining RNA segments or base alteration of the ribonucleotides.

Most mRNA processing relies on typical enzymes consisting of proteins to catalyze the reaction. However, as shown below, more complex RNA processing involves other RNA molecules. These RNAs are involved both in sequence recognition and in the actual chemical reactions of cutting and splicing. In fact, certain introns are self-splicing; that is, they cut themselves out in a reaction that does not require any protein components (see below). Such RNA enzymes are known as ribozymes. As will be discussed in Chapter 13, the formation of peptide bonds during protein synthesis is actually catalyzed by ribosomal RNA, not a protein. The involvement of RNA in such fundamental processes as protein synthesis and RNA processing has led to the idea that ribozymes were more common in early life. Indeed the "RNA world" hypothesis suggests that the original enzymes were all RNA and that protein only assumed this role later in evolution. The RNA world scenario is discussed in more detail in Chapter 26, "Molecular Evolution."

RNA processing sometimes requires other RNA molecules, either as guides or as actual enzymes—ribozymes.

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Processing of RNA

David P. Clark , ... Michelle R. McGehee , in Molecular Biology (Third Edition), 2019

1 RNA Is Processed in Several Ways

RNA is made by RNA polymerase, using a DNA template, in the process known as transcription (see Chapter 11: Transcription). Sometimes the RNA molecule is ready to function immediately after it has been transcribed [e.g., most bacterial messenger RNAs (mRNAs)]. However, in many cases, the RNA needs further processing before it is functional. In these cases, the original RNA molecule, before any further processing occurs, is known as the primary transcript . For specific classes of RNA, the precursor (i.e., primary transcript) may be referred to as pre-mRNA, pre-transfer RNA (tRNA), etc. The term hnRNA (heterogeneous nuclear RNA) was also used previously for the mixture of primary transcripts in the eukaryotic nucleus, before the relationship of precursor RNA to the final processed RNA product was understood.

Many RNA molecules are modified in a variety of ways after being synthesized.

All classes of RNA are subject to processing by base modification and cleavage. In addition, eukaryotic mRNA undergoes capping and tailing as well as splicing (Fig. 12.01). Base modifications occur primarily in tRNA and ribosomal RNA (rRNA), and occur after the RNA is transcribed. These modifications are essential for their proper function in protein translation (see Chapter 13: Protein Synthesis). Certain RNA molecules such as prokaryotic and eukaryotic rRNA are modified by cleavage; that is, the RNA is made as a longer precursor that is trimmed to the correct length. In other related cases, several RNA molecules are included in the same primary transcript, which is then cleaved into several parts. In eukaryotes, the primary transcript for mRNA contains segments called introns or intervening sequences that are not used to encode the final protein product (see Chapter 4: Genes, Genomes, and DNA). Splicing involves the removal of these introns and rejoining of the ends to create a streamlined mRNA with an uninterrupted coding sequence that is translated into a protein.

Figure 12.01. Types of RNA Processing

RNA processing can be divided into cutting/joining RNA segments or base alteration of the ribonucleotides.

Most mRNA processing relies on typical enzymes consisting of proteins to catalyze the reaction. However, as shown later, more complex RNA processing involves other RNA molecules. These RNAs are involved both in sequence recognition and in the actual chemical reactions of cutting and splicing. Such RNA enzymes are known as ribozymes . In fact, certain introns are self-splicing; that is, they cut themselves out in a reaction that does not require any protein components (discussed later). As will be discussed in Chapter 13, Protein Synthesis, the formation of peptide bonds during protein synthesis is actually catalyzed by rRNA, not a protein. The involvement of RNA in such fundamental processes as protein synthesis and RNA processing has led to the idea that ribozymes were more common in early life. Indeed the " RNA world " hypothesis suggests that the original enzymes were all RNA and that protein only assumed this role later in evolution. The RNA world scenario is discussed in more detail in Chapter 29, Molecular Evolution.

RNA processing sometimes requires other RNA molecules, either as guides or as actual enzymes—ribozymes.

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RNA and Protein Synthesis

N.V. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (Second Edition), 2015

RNA Polymerase III Promoters

RNA polymerase III promoters differ significantly from RNA polymerase II promoters in that they are located downstream from the transcription start site and within the transcribed segment of the DNA. For example, in the 5S RNA gene of the South African toad (Xenopus laevis), the promoter is between 45 and 95 nucleotides downstream from the start point. Thus, the binding sites on RNA polymerase III are reversed with respect to the transcription direction, as compared with RNA polymerase II. That is, RNA polymerase II reaches forward to find the start point, and RNA polymerase III reaches backward. In fact, RNA polymerases can slide in either direction along a DNA template; however, they can only synthesize RNA molecules in a 5′→3′ direction.

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Techniques for Nucleic Acid Engineering

Şükrü Tüzmen , ... Candan Hızel , in Omics Technologies and Bio-Engineering, 2018

14.4.1.2.1.1 RNA Gels

RNA is usually run on a formaldehyde agarose gel to highlight the rRNA subunits 28S (upper bright band) and 18S (lower bright band) (Fig. 14.15) (Streit, 2009).

Figure 14.15. Formaldehyde (1%) with RNA samples run at 100   V for 1   hour in 1× MOPS buffer.

 Adapted from http://creativecommons.org/licenses/by-sa/3.0/.

RNA samples are frequently separated on agarose gels that are prepared with formaldehyde as a denaturing agent for the RNA. This chemical serves to limit secondary structure formation (Streit, 2009; Yamanaka, 1997). The gels can be stained using EtBr and visualized under UV light to check the quantity and quality of RNA prior to blotting (Strei, 2009).

RNA separation can also be achieved by PAGE (polyacrylamide gel electrophoresis) containing urea, but oftentimes it is utilized for miRNAs and fragmented RNA (Valoczi, 2004). Usually, an RNA ladder is run next to the samples on an electrophoresis gel to observe the RNA fragment size obtained. However, in total RNA samples, the ribosomal subunits can serve as RNA size markers (Strei, 2009). Because the large ribosomal subunit is 28S (roughly 5   kb), and the small ribosomal subunit is 18S (roughly 2   kb) two noticeable bands will be clearly visible on the gel, the larger band being close to twice the intensity of the smaller one (Streit, 2009; Gortner, 1996).

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