Instruct: Given a question, retrieve documents that can help answer the question.
Query: Why is RNA needed?
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters G, U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.
RNA viruses generally have very high mutation rates compared to DNA viruses, because viral RNA polymerases lack the proofreading ability of DNA polymerases. This is one reason why it is difficult to make effective vaccines to prevent diseases caused by RNA viruses.
Retroviruses also have a high mutation rate even though their DNA intermediate integrates into the host genome (and is thus subject to host DNA proofreading once integrated), because errors during reverse transcription are embedded into both strands of DNA before integration.
Some genes of RNA virus are important to the viral replication cycles and mutations are not tolerated. For example, the region of the hepatitis C virus genome that encodes the core protein is highly conserved, because it contains an RNA structure involved in an internal ribosome entry site.
RNA ladders composed of RNA molecular-weight size markers were initially developed by using the synthetic circle method to produce different-sized markers. This technique was improved upon by inventor Eric T. Kool to use circular DNA vectors as a method for producing RNA molecular-weight size markers. As referred to as the rolling circle method, the improvements of this technique stems from its efficiency in synthesizing RNA oligonucleotides. From the circular DNA template, single-stranded RNA varying in length from 4-1500 bp can be produced without the need for primers and by recycling nucleotide triphosphate. DNA can also be synthesized from the circular template, adding to this technique's versatility. In comparison to runoff transcription, the synthetic circle method produces RNA oligonucleotides without the runoff. In comparison to PCR, the synthetic circle method produces RNA oligonucleotides without the need for polymerase nor a thermal cycler. This method is also cost-efficient in its ability to synthesize grand amounts of product at a lower error rate than machine synthesizers.
Another proposal is that the dual-molecule system we see today, where a nucleotide-based molecule is needed to synthesize protein, and a peptide-based (protein) molecule is needed to make nucleic acid polymers, represents the original form of life.[89] This theory is called RNA-peptide coevolution,[90] or the Peptide-RNA world, and offers a possible explanation for the rapid evolution of high-quality replication in RNA (since proteins are catalysts), with the disadvantage of having to postulate the coincident formation of two complex molecules, an enzyme (from peptides) and a RNA (from nucleotides). In this Peptide-RNA World scenario, RNA would have contained the instructions for life, while peptides (simple protein enzymes) would have accelerated key chemical reactions to carry out those instructions.[91] The study leaves open the question of exactly how those primitive systems managed to replicate themselves — something neither the RNA World hypothesis nor the Peptide-RNA World theory can yet explain, unless polymerases (enzymes that rapidly assemble the RNA molecule) played a role.[91]
Only one of the two DNA strands serve as a template for transcription. The antisense strand of DNA is read by RNA polymerase from the 3' end to the 5' end during transcription (3' → 5'). The complementary RNA is created in the opposite direction, in the 5' → 3' direction, matching the sequence of the sense strand with the exception of switching uracil for thymine. This directionality is because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. This use of only the 3' → 5' DNA strand eliminates the need for the Okazaki fragments that are seen in DNA replication.[1] This also removes the need for an RNA primer to initiate RNA synthesis, as is the case in DNA replication.
RNA viruses can be further classified according to the sense or polarity of their RNA into negative-sense and positive-sense, or ambisense RNA viruses. Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA-dependent RNA polymerase before translation. As such, purified RNA of a positive-sense virus can directly cause infection though it may be less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not infectious by itself as it needs to be transcribed into positive-sense RNA; each virion can be transcribed to several positive-sense RNAs. Ambisense RNA viruses resemble negative-sense RNA viruses, except they also translate genes from the positive strand.
The need for fluorescently tracking RNA rose as its roles in complex cellular functions has grown to not only include mRNA, rRNA, and tRNA, but also RNAi, siRNA, snoRNA, and lncRNA, among others. Spinach is a synthetically derived RNA aptamer born out of the need for a way of studying the role of RNAs at the cellular level. This aptamer was created using Systematic Evolution for Ligands by EXponential enrichment, or SELEX, which is also known as in vitro evolution. The aptamer was designed to be an RNA mimic of green fluorescent protein (GFP); similar to GFP for proteins, Spinach can be used for the fluorescently labeling RNA and tracking it in vivo. A method for inserting the Spinach sequence after an RNA sequence of interest is readily available.
In virology, the term may also be used when referring to mRNA synthesis from an RNA molecule (i.e., RNA replication). For instance, the genome of a negative-sense single-stranded RNA (ssRNA -) virus may be template for a positive-sense single-stranded RNA (ssRNA +). This is because the positive-sense strand contains the information needed to translate the viral proteins for viral replication afterwards. This process is catalyzed by a viral RNA replicase.[2]
Negative sense ssRNA viruses need RNA polymerase to form a positive sense RNA. The positive-sense RNA acts as a viral mRNA, which is translated into proteins for the production of new virion materials. With the newly formed virions, more negative sense RNA molecules are produced.
At first, regulatory RNA was thought to be a eukaryotic phenomenon, a part of the explanation for why so much more transcription in higher organisms was seen than had been predicted. But as soon as researchers began to look for possible RNA regulators in bacteria, they turned up there as well.[41] Currently, the ubiquitous nature of systems of RNA regulation of genes has been discussed as support for the RNA World theory.[40][48] Bacterial small RNAs generally act via antisense pairing with mRNA to down-regulate its translation, either by affecting stability or affecting cis-binding ability.[40] Riboswitches have also been discovered. They are cis-acting regulatory RNA sequences acting allosterically. They change shape when they bind metabolites so that they gain or lose the ability to bind chromatin to regulate expression of genes.[49][50]
Positive-sense (5' to 3') viral RNA signifies that a particular viral RNA sequence may be directly translated into the desired viral proteins. Therefore, in positive-sense RNA viruses, the viral RNA genome can be considered viral mRNA, and can be immediately translated by the host cell. Unlike negative-sense RNA, positive-sense RNA is of the same sense as mRNA. Some viruses (e.g., Coronaviridae) have positive-sense genomes that can act as mRNA and be used directly to synthesize proteins without the help of a complementary RNA intermediate. Because of this, these viruses do not need to have an RNA polymerase packaged into the virion.
A negative-sense single-stranded RNA virus (or (-)ssRNA virus) is a virus that uses negative sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The negative viral RNA is complementary to the mRNA and must be converted to a positive RNA by RNA polymerase before translation. Therefore, the purified RNA of a negative sense virus is not infectious by itself, as it needs to be converted to a positive sense RNA for replication. These viruses belong to Group V on the Baltimore classification.
Damha and his research group have also made several contributions to the synthesis of RNA and novel RNA structures. These include the synthesis of branched and lariat RNA structures as well as novel acetal levulinyl ester (ALE)-based protecting group strategies that allow RNA to be synthesized directly on glass microarrays (RNA "chips") by eliminating the need for fluoride-mediated deprotection.
The function of sbRNAs may therefore be similar to that of vertebrate Y RNAs, namely acting as part of the Ro-RNA particle to control RNA quality and playing a role in chromosomal replication. Deletion of sbRNA does not prevent chromosome replication in "C. elegans", but this may be a result of other sbRNAs substituting missing elements (as in human Y RNA). This theory also explains why RNAi studies failed to detect a phenotype for knocked out sbRNAs.
The mechanism of the editosome involves an endonucleolytic cut at the mismatch point between the guide RNA and the unedited transcript. The next step is catalyzed by one of the enzymes in the complex, a terminal U-transferase, which adds Us from UTP at the 3' end of the mRNA. The opened ends are held in place by other proteins in the complex. Another enzyme, a U-specific exoribonuclease, removes the unpaired Us. After editing has made mRNA complementary to gRNA, an RNA ligase rejoins the ends of the edited mRNA transcript. As a consequence, the editosome can edit only in a 3' to 5' direction along the primary RNA transcript. The complex can act on only a single guide RNA at a time. Therefore, a RNA transcript requiring extensive editing will need more than one guide RNA and editosome complex.
Gene expression first involves transcription, in which DNA is used as a template to produce RNA. In the case of genes encoding proteins, that RNA produced from this process is messenger RNA (mRNA), which then needs to be translated by ribosomes to form a protein. As ribosomes are located outside the nucleus, mRNA produced needs to be exported.[56]
If the RNA world existed, it was probably followed by an age characterized by the evolution of ribonucleoproteins (RNP world),[2] which in turn ushered in the era of DNA and longer proteins. DNA has better stability and durability than RNA; this may explain why it became the predominant storage molecule.[10] Protein enzymes may have come to replace RNA-based ribozymes as biocatalysts because their greater abundance and diversity of monomers makes them more versatile. As some co-factors contain both nucleotide and amino-acid characteristics, it may be that amino acids, peptides and finally proteins initially were co-factors for ribozymes.[8]
Some caution needs to be applied in the interpretation of trans-splicing events detected in high-throughput sequencing experiments as the reverse transcriptase enzymes ubiquitously used to determine RNA sequences are capable of introducing apparent trans-splicing events that were not present in the original RNA. Some chimeric RNAs have been confirmed by other methods however.
Transcription of rRNA yields a long precursor molecule (45S pre-rRNA) which still contains the ITS and ETS. Further processing is needed to generate the 18S RNA, 5.8S and 28S RNA molecules. In eukaryotes, the RNA-modifying enzymes are brought to their respective recognition sites by interaction with guide RNAs, which bind these specific sequences. These guide RNAs belong to the class of small nucleolar RNAs (snoRNAs) which are complexed with proteins and exist as small-nucleolar-ribonucleoproteins (snoRNPs). Once the rRNA subunits are processed, they are ready to be assembled into larger ribosomal subunits. However, an additional rRNA molecule, the 5S rRNA, is also necessary. In yeast, the 5S rDNA sequence is localized in the intergenic spacer and is transcribed in the nucleolus by RNA pol.
As RNA integrity has long been known to be a problem in molecular biology studies, there are a few methods that have been used historically to determine the integrity of RNA. The most popular has long been agarose gel electrophoresis with ethidium bromide staining, allowing one to visualize the bands from the rRNA peaks. The height of the 28S and 18S bands can be compared to each other, with a 2:1 ratio indicating non-degraded RNA. While this method is very cheap and easy, there are several issues with this method, primarily its subjectivity, leading to inconsistent, non-standardized RNA quality assessments, and the large amounts of RNA that are needed to visualize it on an agarose gel, which can be problematic if there is not much RNA to work with. There are also a number of different problems that can arise from agarose gel electrophoresis, such as poor loading, uneven running, and uneven staining that lead to increased variability in the accuracy of using agarose gel electrophoresis to determine RNA integrity. Clearly, a standardized, repeatable, objective method of determining RNA integrity was needed.
The RNA recognition motif, which is the most common RNA-binding motif, is a small protein domain of 75–85 amino acids that forms a four-stranded β-sheet against the two α-helices. This recognition motif exerts its role in numerous cellular functions, especially in mRNA/rRNA processing, splicing, translation regulation, RNA export, and RNA stability. Ten structures of an RRM have been identified through NMR spectroscopy and X-ray crystallography. These structures illustrate the intricacy of protein–RNA recognition of RRM as it entails RNA–RNA and protein–protein interactions in addition to protein–RNA interactions. Despite their complexity, all ten structures have some common features. All RRMs' main protein surfaces' four-stranded β-sheet was found to interact with the RNA, which usually contacts two or three nucleotides in a specific manner. In addition, strong RNA binding affinity and specificity towards variation are achieved through an interaction between the inter-domain linker and the RNA and between RRMs themselves. This plasticity of the RRM explains why RRM is the most abundant domain and why it plays an important role in various biological functions.
As retroviral particles consistently have similar amounts of RNA, lack of genomic RNA in some viral particles must be supplemented by cellular RNA. When treated with RNase, retoviral cores are disrupted. Therefore, regardless of whether the NC domain of Gag needs assistance in assembling retroviruses, the RNA encapsidation (packaging) signal does ensure the presence of RNA material in the viral particles, which serve as important structural elements.
The Pseudomonadales-1 RNA motif is a conserved RNA structure that was discovered by bioinformatics.
The Pseudomonadales-1 motif often exhibits an apparent sarcin-ricin loop, a type internal loop common in RNA.
Pseudomonadales-1 motif RNAs are found in relatively closely related species of Pseudomonadales. Despite this narrow distribution, the Pseudomonadales-1 RNA motif does not exhibit many invariant nucleotide positions, suggesting that it does not need to be highly conserved at the primary sequence level.
One caveat of MS2 tagging is that many copies of the MS2 stem-loop inside the RNA need to be added to produce enough signal to view and track one RNA molecule in the nucleus. When tracking more than one RNA sequence in the nucleus of cultured cells, more than one target sequence is needed. This could be affected by the MS2 protein, which has a classical basic nuclear localization signal (NLS), so it could affect the location of the RNA complex, and the nucleus would have most of the GFP-MS2 (Robert Singer’s lab). The accumulation of GFP-MS2 in the nucleus will result in strong nuclear fluorescence signals, which will delay or prevent the analysis of RNA nuclear localization because it will hinder the analysis of splicing, RNA editing, the nuclear export of RNA, and RNA translation. Moreover, due to the addition of the tag, the RNA secondary structure may introduce an artifact.
On January 20, Lt. General Katawal, Royal Nepalese Army (RNA) Chief of General Staff, told Ambassador James F. Moriarty that he was worried that the government did not have a unified strategy for tackling the Maoist insurgency. Katawal reiterated that there was no purely military solution to the insurgency; the RNA could only work to create space for a negotiated political solution. He noted that the civilian government should lead the effort to inform its citizens about Nepal's overall plan to tackle the insurgency. Katawal stressed that a civilian government needed to explain to the people what the military was doing and why, especially why the government needed to restrict civil liberties. 