RNA: Introduction, Synthesis and Types

Read this article to learn about the synthesis process and types of RNA. The three classical types of RNA are:

(1) Messenger RNA (2) Ribosomal RNA and (3) Transfer RNA. Besides this three types of RNA, new types of RNA molecules are discovered which are involved in various activities directly or modulate the activity. They are: (1) Non-Coding RNA (2) Small Nuclear RNA (3) Small Nucleolar RNA (4) Short Interfering RNAs (5) Micro-RNAs (6) Ribozymes (7) XISTRNA (8) Double-Stranded RNA and (9) RNA Secondary Structures.

Introduction:

RNA is a polymer of ribo-nucleoside-phosphates. Dr. Severo Ochoa discovered the RNA and got 1959 Nobel Prize for Medicine. The sequence of the 77 nucleotides of yeast RNA was found by Robert W. Holley in 1964, winning Holley the 1968 Nobel Prize for Medicine. In 1976, Walter Fiers and his team at the University of Ghent determined the complete nucleotide sequence of bacteriophage MS2-RNA.

Its backbone is comprised of alternating ribose and phosphate groups. Ribose is a five carbon sugar with carbons numbered 1’ through 5′. A base is attached to the Y position, generally adenine (A), cytosine (C), guanine (G) or uracil (U). Adenine and guanine are purines, cytosine and uracil are pyrimidine’s. A phosphate group is attached to the 3′ position of one ribose and the 5′ position of the next. Most cellular RNA is single stranded, although some viruses have double stranded RNA (Fig. 5.1).

Several other bases are occasionally found in RNAs including: thymine, pseudouridine and methylated cytosine and guanine. However, there are also numerous modified bases and sugars found in RNA that serve many different roles. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C-N bond to a C-C bond, and ribothymidine (T), are found in various places (most notably in the TΨC loop of tRNA). Thus, it is not technically correct to say that uracil is found in RNA in place of thymine. Another notable modified base is hypoxanthine (a deaminated Guanine base whose nucleoside is called Inosine).

Inosine plays a key role in the Wobble Hypothesis of the Genetic Code. There are nearly 100 other naturally occurring modified nucleosides, of which pseudouridine and nucleosides with 2′-0-methylribose are by far the most common. The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that in ribosomal RNA, many of the post-translational modifications occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function. The single RNA strand is folded upon itself, either entirely or in certain regions.

In the folded region a majority of the bases are complementary and are joined by hydrogen bonds. This helps in the stability of the molecule. In the unfolded region the bases have no complements. Because of this RNA does not have the purine pyrimidine equality that is found in DNA.

Inside of cells, there are three major types of RNA: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). There are a number of other types of RNA present in smaller quantities as well, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and the 4.5S signal recognition particle (SRP) RNA. Novel species of RNA continue to be identified. RNA serves a multitude of roles in living cells.

These include: serving as a temporary copy of genes that is used as a template for protein synthesis (mRNA), functioning as adaptor molecules that decode the genetic code (tRNA) and catalyzing the synthesis of proteins (rRNA). There is much evidence implicating RNA structure in biological regulation and catalysis. Interestingly, RNA is the only biological polymer that serves as both a catalyst (like proteins) and as information storage (like DNA).

For this reason, it has be postulated RNA, or an RNA-like molecule, was the basis of life early in evolution. RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions of ribosomes, and acts as an essential carrier molecule for amino acids to be used in protein synthesis.

Synthesis of RNA:

There are three types of RNA; messenger RNA (mRNA) or template RNA, ribosomal RNA (rRNA) and soluble RNA (sRNA) or transfer RNA (tRNA). Ribosomal and transfer RNA comprise about 98% of all RNA. All three forms of RNA are made on a DNA template. Transfer RNA and messenger RNA are synthesized on DNA templates of the chromosomes, while ribosomal RNA is derived from nucleolar DNA.

The three types of RNA are synthesized during different stages in early development. Most of the RNA synthesized during cleavage is mRNA. Synthesis of tRNA occurs at the end of cleavage, and rRNA synthesis begins during gastrulation.

Synthesis of RNA is usually catalyzed by an enzyme RNA polymerase using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found “upstream” of a gene).

The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3′ to 5′ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5′ to 3′ direction.

The DNA sequence also dictates where termination of RNA synthesis will occur. RNAs are often modified by enzymes after transcription. For example, a poly (A) tail and a 5′ cap are added to eukaryotic pre-mRNA and introns are removed by the splice some.

There are also a number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material.

Types of RNA:

A. Messenger RNA (mRNA):

Messenger RNA (mRNA) is only 5-10% of total RNA present in the cell. In 1961, Francis Jacob and Jacques Monod for the first time proposed the name mRNA. This RNA carries genetic information from DNA to the ribosome. Since mRNA is transcribed on DNA (genes), its base sequence is complementary to that of the segment of DNA on which it is transcribed. Usually each gene transcribes its own mRNA. Therefore, there are approximately as many types of mRNA molecules as there are genes.

There may be 1,000 to 10,000 different species of mRNA in a cell. These mRNA types differ only in the sequence of their bases and in length. Messenger RNA is first synthesized by genes as nuclear heterogeneous RNA (hnRNA), being so called because hnRNAs varies enormously in their molecular weight as well as in their nucleotide sequences and lengths, which reflects the different proteins they are destined to code for translation.

The coding sequence of the mRNA determines the amino acid sequence in the protein. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns i.e., non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA.

In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain period of time the RNA degrades into its component nucleotides with the assistance of ribonucleases. So, mRNA is short lived. When one gene (cistron) codes for a single mRNA strand the mRNA is said to be monocistronic. In many cases, however, several adjacent cistrons may transcribe an mRNA molecule, which is then said to be polycistronic or polygenic (e.g., in prokaryotes). This polycistronic mRNA encode multiple proteins that are separately translated from the same mRNA molecule (Table 5.2).

The eukaryotic mRNA is typically monocistronic i.e., only one species of polypeptide chain is translated per mRNA molecule. Most mRNAs contain a significant non-coding segment, that is, a portion that does not direct the assembly of amino acids. For example, approx. 25% of each globin mRNA consists of non-coding, non-translated regions. Non-coding portions are found on both the 5′ and 3′ ends of a messenger RNA and contain sequences that have important regulatory roles.

Messenger RNA is always single stranded. It contains mostly the bases adenine, guanine, cytosine and uracil. There are few unusual substituted bases. Although there is a certain amount of random coiling in extracted mRNA, there is no base pairing. In fact base pairing in the mRNA strand destroys its biological activity. It is heterogeneous in size and sequence. The cap (5′-G) is added to the mRNA after transcription.

The addition of 5′ G is catalyzed by a nuclear enzyme, guanylyl transferase. The cap is linked to the 5′ terminus of the mRNA through an unusual 5′, 5’- triphospahe linkage. The 5′ cap is formed by condensation of a molecule of GTP with the triphosphate at the 5′ end of the transcript. The guanine is subsequently methylated at N-7 to form 7-methylguanosine. Additional methyl groups are added to the 2′ hydroxyls (—OH) of the first and second nucleotides adjacent to the cap. The methyl groups are derived from S-adenosylmethionine. This cap serves to identify this RNA molecule as an mRNA to the translational machinery.

In addition, most mRNA molecules contain a poly-Adenosine tail (poly ‘A’ tail) at the 3′ end. Both the 5′ cap and the 3′ tail are added after the RNA is transcribed and contribute to the stability of the mRNA in the cell. Therefore, the molecular weight of mRNAs varies from some hundreds to Thousands of Daltons.

The molecular weight of an average sized mRNA molecule is about 500,000, and its sedimentation coefficient is 8S. mRNA varies greatly in length and molecular weight. Since most proteins contain at least a hundred amino acid residues, mRNA must have at least 300 (100X3) nucleotides on the basis of the triplet code. In E. coli the average mRNA strand has 900 to 1,500 nucleotide units which would code polypeptide chains of 300-500 amino acids. Molecules containing 12,000 nucleotide units are also known.

The structure of prokaryotic and eukaryotic mRNA is as follows:

1. 5′ Cap:

At the 5′ end of the mRNA molecule in most eukaryote cells and animal virus molecules is found a ‘cap’. This cap is formed by the methylation of any of the four nucleotides. The cap helps mRNA to bind to the ribosomes. Without the cap mRNA molecules bind very poorly to the ribosomes. The bacterial mRNA does not have 5’cap. But they have specific ribosome binding site about six nucleotide long, which occurs at several places in the mRNA molecules. These are located at four nucleotides upstream from AUC.

2. Non-coding Regions:

As the name indicates these regions do not code for protein. There are two non-coding regions. First non-coding region (NCI) is followed by a 5’cap and is 10 to 100 nucleotides in length. This region is rich in A and U residues. The second non- coding region (NC2) is followed by termination codon and is 50-150 nucleotides long and contains an AAUAAA residues.

3. Initiation Codon:

Initiation codon is AUG in both prokaryotes and eukaryotes. Bacterial ribosomes bind directly to the AUG region of the mRNA to start the protein synthesis, whereas this is not there in the case of eukaryotes.

4. The Coding Region:

It consists of about 1,500 nucleotides. This region is responsible for coding protein with several ribosomes. The combination of mRNA strand with several ribosomes is called polyribosomes.

5. The Termination Codon:

The termination codon is required to give the signal to stop protein synthesis.

6. The poly (A) Sequence:

The non-coding region II is followed by poly (A) sequence in the eukaryotic mRNA. The prokaryotic mRNAs lack poly (A). The poly (A) sequences of 200-250 nucleotides are present at 3’OH end of mRNA. Poly (A) sequences are added when mRNA is present inside the nucleus. The function of poly (A) sequence in translation is unknown (Fig. 5.3).

Difference between prokaryotic and eukaryotic mRNA:

Prokaryotic mRNA:

1. Translation begins when the mRNA is still being transcribed on DNA.

2. Prokaryote mRNA are very short lived. It constantly under goes breakdown to its constituent ribonucleotides by ribonucleases.

3. In Prokaryotic mRNA are polycistronic.

4. The mRNA undergo very little processing after being transcribed.

5. Prokaryotic mRNA do not have poly (A) tail.

Eukaryotic mRNA:

1. Translation begins when the transcription is Completed.

2. Eukaryotic nRNAs are long lived. Thus are metabolically stable.

3. In Eukaryotic  mRna are monocistronic .

4. The mRNA undergoes several processing after being transcribed such as polyadenylation capping and methylation.

5. Eukaryotic mRNA have poly (A) tail.

B. Ribosomal RNA (rRNA):

Ribosomal RNA is extremely abundant and makes up to 80% of RNA found in a typical eukaryotic cytoplasm. Ribosomal RNA consists of a single strand twisted upon itself in some regions. It has helical regions connected by intervening single strand regions. The helical regions may show presence or absence of positive interaction. In the helical region most of the base pairs are complementary, and are joined by hydrogen bonds. In the unfolded single strand regions the bases have no complements.

In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. Ribosomal RNA (rRNA) is a component of the ribosomes. The base sequence of rRNA is complementary to that of the region of DNA where it is synthesized. Ribosomal RNA is formed from only a small section of the DNA molecule, and hence there is no definite base relationship between rRNA and DNA as a whole.

Primarily there are two types of ribosomes, one is 70s (prokaryotes) and the other is 80s (eukaryotes). The 70S ribosome of prokaryotes consists of a 30S subunit and a 50S subunit. The 30S subunit contains 16S rRNA, while the 50S subunit contains 23S and 5S rRNA. The 80S eukaryote ribosome consists of a 40S and a 60S subunit.

In vertebrates the 40S subunit contains 18S rRNA, while the 60S subunit contains 28-29S, 5.8S and 5S rRNA. In plants and invertebrates, the 40S subunit contains 16-18S RNA, while the 60S subunit contains 25S and 58 and 5.8S rRNA.

C. Transfer or tRNA:

The tRNA molecules are key to the translation process of the mRNA sequence into the amino acid sequence of proteins (at least one type of tRNA for every amino acid). To be precise, the amino-acyl-tRNA-synthase proteins are the ‘true’ translators of the genetic code into an amino acid sequence. These synthetases acetylate tRNA molecules with the proper amino acid that corresponds to the anti-codon in the structure of the tRNA molecule.

The anti-codon later recognizes the codon, the triple base sequence which ‘codes’ for the amino acid along the mRNA strand. A failure of properly acetylating the tRNA with the right amino acid results in an amino acid mutation even though the DNA sequence has not been changed. tRNA molecules are small nucleic acids of 60- 95 nucleotides, mostly 76, with a molecular weight 18-20kD, with the secondary structure resembling a clover leaf. Here are a few common features shared by all tRNA molecules found in various organisms.

(1) 5′ terminus is always phosphorylated.

(2) 7 bp stem, may have non-Watson & Crick pairing (like GU) acceptor or amino acid stem at 3′ terminus in which last three nucleotides are CCA-3′-OH. These are added after transcription and amino acylation occurs at 3′-OH group of last base ‘A’.

(3) 3-4 bp stem and loop contains the base dihydrouridine (D) [D- arm.]

(4) 5 bp stem and loop containing anti-codon triplet [anti-codon arm].

(5) 5 bp stem and loop contains sequence T C, standing for [T- arm] pseudouridine.

(6) Variable arm (between anti-codon and T-arm) length, 3-21 nucleotides.

(7) Contains numerous modified bases (up to 25%) which are all post-transcriptionally modified.

The three dimensional structure of tRNA resembles an L-shaped molecule with the D-arm and anti-codon loop building one stretch and the T-arm and acceptor stem building the other stretch being deposed by ~90 to one another (interstem angle of 82 by X-ray refinement and 92 in an electron microscopy study). The molecule is about 6 nm in each direction with the anti-codon to acceptor 3′-term ends being 7.6 nm apart. The diameter of both arms is about 2.0 to 2.5 nm.

The structural complexity of tRNA is reminiscent of that of a protein with 71 out of 76 bases participating in stacking interaction (of which 42 in double helical stem structures). 9 bp interactions are cross linking the tertiary structure, i.e., they interact with bases from a different stem and loop region. All of these 9 bp are non-Watson-Crick associations and are highly conserved which makes it likely to predict similar structures for all tRNA molecules (in fact, only few tRNA molecules have been crystallized and their structure determined) (Fig. 5.4).

New Types of RNA:

Besides mRNA, tRNA and rRNA, the three classically known RNA molecules, new types of RNA molecules are discovered in recent years, which are involved in various activities directly or they modulate the activity.

Some of the new types of RNA which are involved in various activities directly are as follows:

1. Non-Coding RNA:

RNA genes (sometimes referred to as non-coding RNA or small RNA) are genes that encode RNA that is not translated into a protein. The most prominent examples of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found, and thus RNA genes may play a much more significant role than previously thought.

In the late 1990s and early 2000, there has been persistent evidence of more complex transcription occurring in mammalian cells (and possibly others). This could point towards a more widespread use of RNA in biology, particularly in gene regulation. A particular class of non-coding RNA, micro RNA, has been found in many metazoans (from Caenorhabditis elegans to Homo sapiens) and clearly plays an important role in regulating other genes.

2. Small Nuclear RNA (snRNA):

About a dozen genes for snRNAs have been described, each present in multiple copies in the genome. Small nuclear RNAs combine with certain U-proteins to form snRNPs. These executive molecules have roles in editing other classes of RNA. The “U” designation was given to the snRNAs because they were found to be rich in uridylic acid.

An important example is the small ribonuclear proteins (snRNPs) that are components of the spliceosomes. Spliceosomes edits introns nitrogen base sequences out of pm RNA forming mRNA. U6 is transcribed in the nucleoplasm by RNA polymerase III, while U1, U2, U4 and U5 are transcribed by RNA polymerase II. The snRNPs are involved in processing of pmRNA.

3. Small Nucleolar RNA (snoRNA):

In eukaryotic cells, rRNA and snRNA are extensively modified and processed in the nucleolus. Much of this activity is affected by snoRNAs. It appears that the coding for snoRNAs lies in introns and other intergenic regions (non-protein coding). Small nucleolar RNA can be considered to be a subgroup of the snRNAs but should not be confused with the snRNAs mediating mRNA splicing, i.e. the spliceosomal RNAs.

It is interesting to note that snoRNAs have not been found in any prokaryotes. Many snoRNAs and snRNAs have been found to be encoded in introns. It has been suggested that snoRNAs are possibly processed from the out-spliced introns by exonucleases.

4. Short Interfering RNAs (siRNA):

Short interfering RNAs and miRNAs were discovered in different works, but their biogenesis and assembly into RNA-protein complexes and their function in down regulating gene expression are closely related. Short interfering RNAs and mi RNAs share common RNAse III processing enzyme, the dicer enzymes and closely related effector complexes for post-transcriptional repression of protein synthesis. On the other hand, siRNAs and miRNAs differ in their molecular origins. In the cytoplasm the dicer enzymes split the dsRNA primer molecules. The finished siRNAs in animals are usually 21-22 nitrogen bases long, similar to miRNAs.

5. Micro-RNAs (miRNAs):

Micro-RNAs are a class of small, non-coding RNAs that regulate gene expression in a sequence specific manner as required in embryonic development. Micro-RNAs have been found throughout diverse eukaryotes genomes including plants. They can inhibit protein expression by shutting off translation or by targeting mRNA for degradation. Micro-RNAs were first discovered in 2001 in the widely studied worm Caenorhabdtis elegans.

Micro-RNAs genes produce short (~22 nitrogen bases) ss segments that fold over on themselves forming a short section of dsRNA in hairpin like structure. Humans express over 460 genetically encoded miRNAs. These miRNAs make up more than 1 % of human genome and may regulate over 30% of all protein coding genes. Micro-RNAs can pair exactly with a mRNA and cause its cleavage and destruction, or it can pair partially with mRNA and produce translational inhibition (block the ribosome). It is presumed that they are involved in regulating development by controlling as transcriptional factor.

6. Ribozymes:

Ribozymes are catalytic RNA enzymes that act to alter covalent structure in other classes of RNAs and certain molecules. They occur in ribosomes, nucleus and chloroplasts of eukaryotic organisms. Some viruses including several bacteriophages also have ribozymes. An optimum concentration of metal ions such as Mg⁺⁺ and K⁺ is associated with their effective functioning. Ribozymes generally act as molecular scissors cutting precursor RNA molecules at specific sites. Surprisingly, they also serve as molecular staplers, which ligate or join two RNA molecules together.

Ribozymes are involved in the transformation of large precursor molecules of tRNA, rRNA and mRNA into smaller final products. In their active form, ribozymes are complexed with protein molecules, e.g., the enzyme ribonuclease-P (RNAse-P) is found in all living organisms. Ribonuclease-P is a heterodimer containing one molecule of protein and one molecule of RNA.

This ribozyme cleaves the head 5′ end of the precursors to the tRNAs. These enzymes are involved in autocatalytic splicing of preRNA making contiguous RNA. This cleavage is carried out by RNA part of the heterodimer.

Synthetic ribozymes can be used in genetic engineering applications to stop the expression of any gene in a sequence-specific manner and therefore may be useful in cancer therapy and HIV treatment. Ribozymes can be inserted into the cells has synthetic oligonucleotides with a gene gun or the cell can synthesize them itself with engineered genes.

7. XISTRNA:

XIST stands for X-inactive-specific transcript. It is a large (~ 17kb) RNA coded by a gene on the X chromosome (8 exons are involved with human xist). xistRNA accumulates in female somatic cells along the X chromosome containing the active xist gene and proceeds to inactivate nearly all the 100s of genes on that X chromosome. The xistRNA does not travel over to any other chromosome in nucleus. The barr body seen in the cell nucleus with light microscopy is inactive X chromosome covered with xistRNA. The entire xistRNA X-blocking mechanism is very complex.

8. Double-Stranded RNA (dsRNA):

Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all ‘higher’ cells. dsRNA forms the genetic material of some viruses. In eukaryotes, it acts as a trigger to initiate the process of RNA interference and is present as an intermediate step in the formation of siRNAs (small interfering RNAs). siRNAs are often confused with miRNAs; siRNAs are double-stranded, whereas miRNAs are single-stranded.

Although initially single stranded, there are regions of intra-molecular association causing hairpin structures in pre-miRNAs. Very recently, dsRNA has been found to induce gene expression at transcriptional level, a phenomenon named “small RNA induced gene activation (RNAa)”. Such dsRNA is called “small activating RNA (saRNA)”.

9. RNA Secondary Structures:

The functional form of single stranded RNA molecules (like proteins) frequently requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements which are hydrogen bonds within the molecule. This leads to several recognizable “domains” of secondary structure like hairpin loops, bulges and internal loops.

The secondary structure of RNA molecules can be predicted computationally by calculating the minimum free energies (MFE) structure for all different combinations of hydrogen bonding’s and domains. There has been a significant amount of research directed at the RNA structure prediction problem.