RNA Types and Structure

General Features of RNA

Structure

Ribonucleic acid (RNA) is a single-stranded polymer of nucleotides that are linked through 3’–5’ phosphodiester bonds.

Nucleotides are the basic unit of any nucleic acid. The nucleotides in RNA are formed by the following parts:

1. Ribose (sugar): Carbon 2’ is bonded to hydroxyl (OH). 
   Note: In deoxyribonucleic acid (DNA), the word deoxyribose means that there is no oxygen (O) attached to carbon 2’.
2. Phosphate group: bonded to the carbon 5’ of ribose; links to the OH of the carbon 3’ of the next nucleotide, forming a phosphodiester bond
3. Nitrogenous base: bonded to the carbon 1’ of ribose
   • Purine bases:
     • Adenine
     • Guanine
   • Pyrimidine bases:
     • Cytosine
     • Uracil (Note: Uracil differs from DNA, which has thymine.)

Nucleic acid components:

• Nucleobase: nitrogenous base (adenine)
• Nucleoside: nitrogenous base + sugar (adenosine)
• Nucleotide: nitrogenous base + sugar + phosphate (adenosine monophosphate)
• Nucleic acid: polymer of nucleotide (RNA)

Function

• Protein synthesis (messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA))
• Gene expression regulation (micro RNA (miRNA), small regulatory RNA (sRNA), small interfering RNA (siRNA))
• Processing of other RNAs (small nuclear RNA (snRNA), small nucleolar RNA (snoRNA))
• Catalysis or metabolic function (ribozymes)
• Genetic material in some viruses
• Genome defense (siRNA (RNA interference), PIWI-interacting RNA (piRNA) in eukaryotes, CRISPR in prokaryotes)

Coding versus non-coding RNA

• Coding RNA (translated into protein) such as:
  • mRNA
  • RNA of the viral genome
• Non-coding RNA (other roles in the cell) such as:
  • tRNA
  • rRNA
  • miRNA
  • snoRNA
  • snRNA

Messenger RNA (mRNA)

Structure

• Coding DNA sequence (CDS):
  • Broken into 3 base sequences known as codons (see mnemonic); correspond to specific amino acids
  • Begins with an initiation codon (AUG)
  • Ends with a termination codon (UGA, UAA, and UAG)
• Untranslated region (UTR) or non-coding sequences (see differences below):
  • 5’ cap
  • 3’ poly-A tail
  • Shine-Dalgarno sequence (in prokaryotes)

Mnemonic

To remember mRNA start codons, use the mnemonic AUG:

• InAUGurates protein synthesis 
• Are U Going

To remember mRNA stop codons, use the mnemonics UGA, UAA, and UAG:

• U Go Away
• U Are Away
• U Are Gone

Function

Messenger RNA (mRNA) serves as an amino acid template for protein synthesis.

• During transcription, mRNA has the following characteristics:
  • Complementary to the antisense or template strand (facilitated by the enzyme RNA polymerase)
  • Becomes a copy of the coding strand (sense strand)
• Messenger RNA is synthesized from the 5’ end to the 3’ end (because RNA polymerase can only add nucleotides to the 3’ end of the mRNA chain).
• The initial transcript is known as heterogeneous nuclear RNA (hnRNA).
• Processed hnRNA, which has the addition of the 5’ cap and the 3’ poly-A tail followed by splicing, becomes mRNA.

Prokaryotic versus eukaryotic mRNA

Prokaryotic mRNA

• Polycistronic: 
  • One mRNA = several polypeptides 
  • In bacteria, clusters of related genes or operons are transcribed together in a single mRNA.
• The CDS is translated into proteins immediately after being synthesized (no post-transcriptional modifications).
• The 5’ UTR contains a Shine-Dalgarno sequence that helps recruit the ribosome to mRNA for protein synthesis.

Eukaryotic mRNA

• Monocistronic: 
  • One mRNA = single polypeptide 
  • One initiation and termination codon producing one polypeptide chain
• Modified extensively before being translated into proteins:
  • Capping of 5’ end: linkage of 7-methylguanosine 
    • Aids in recognition by the protein synthesis machinery 
    • Protects from degradation by exonucleases
  • The CDS undergoes modification or splicing of introns (non-coding segments).
  • Addition of the 3’ poly-A tail: chain of adenylate molecules (maintains the stability of mRNA as it exits the nucleus into the cytosol)

There is a long-held belief that prokaryotes lack the 5’ cap. Recently, however, some bacteria have been found to have a 5’ nicotinamide adenine dinucleotide (NAD) cap:

• Appears to protect bacterial RNA from degradation
• The 5’ NAD cap found in some eukaryotes actually promotes decay rather than provide protection.
• 5’ 7-methylguanosine cap of eukaryotes: added post-transcription
• 5’ NAD cap in prokaryotes and eukaryotes: added in initiation step of transcription

Transfer RNA (tRNA)

Structure

• Transfer RNA is a single polynucleotide made up of an average of 75 nucleotides.
• Has modified bases such as inosine, dihydrouridine, and pseudouridine
• The distinctive fold creates a 2-dimensional shape resembling a cloverleaf.
• The 3-dimensional or tertiary structure of tRNA is actually L shaped.
• Parts of tRNA consist of:
  • Acceptor stem:
    • Contains the 3’ CCA (cytosine-cytosine-adenine) sequence: 
      • Amino acid attachment site 
      • Forms a covalent bond to a specific amino acid via aminoacyl-tRNA synthetase
    • The acceptor stem also contains parts of the 5’ end of tRNA.
  • Anti-codon loop:
    • The 3-base sequence is complementary to the mRNA triplet code for the specific amino acid.
  • D-arm (contains dihydrouridine) and T-arm (pseudouridine loop) or TΨC arm (thymidine-pseudouridine-cytidine):
    • The D-arm is believed to be a recognition site for aminoacyl-tRNA synthetase.
    • The T-arm aids in ribosome attachment.
  • Variable loop:
    • May or may not be present; allows for further classification of tRNA

Function

Transfer RNA transports amino acids to ribosomes for assembly into proteins. This process is carried out by these 2 main actions:

• Chemically links to a specific amino acid:
  • Specificity is due to the enzyme aminoacyl-tRNA synthetase: 
    • Each enzyme recognizes only one amino acid and the corresponding tRNA.
    • 20 aminoacyl-tRNA synthetase enzymes correspond to each of the 20 amino acids.
    • Uncharged tRNA: no amino acid attached
    • Charged tRNA: amino acid attached
  • Aminoacyl-tRNA synthetase links the amino acid to the 3’ terminal of the tRNA acceptor stem. 
• Forms base pairs with the codon in the mRNA:
  • Via tRNA anti-codon loop
  • Determines the amino acid to carry to the ribosome for protein assembly
  • One tRNA carries one specific amino acid but one tRNA can read more than one codon.
  • More than one codon can code for one amino acid (codon degeneracy):
    • Example: 1 tRNA = 1 amino acid (Phe) = different codons, but the same first 2 bases: UUU, UUC
  • The first 2 codon bases: main determining bases for amino acids
  • Between the 3rd codon base and the complementary 1st anti-codon base of the tRNA, more than one base pair is possible: 
    • Can follow atypical base-pairing (wobbling)
    • Example: Inosine (I), a modified base found in tRNA, pairs with the uracil (U), adenine (A), and cytosine (C).

Ribosomal RNA (rRNA)

Structure

Ribosomes consist of 2 subunits of unequally sized rRNA, and the sizes are measured in terms of an “S” value (Svedberg or sedimentation unit):

• Based on a measure of sedimentation velocity in a centrifuge (higher “S” value = faster sedimentation = greater mass)
• Therefore, “S” values are not additive.
• Prokaryotic ribosomes: 70S consisting of 50S (large subunit) and 30S (small subunit)
• Eukaryotic ribosomes: 80S consisting of 60S (large subunit)  and 40S (small subunit)
• Has 3 functionally distinct tRNA binding sites:
  • Amino acyl (A) site (accepts incoming aminoacyl tRNA)
  • Peptidyl (P) site (for the peptidyl tRNA to which the growing peptide chain is attached)
  • Exit (E) site (where the deacylated tRNA exits the ribosome)

Function

• Most abundant form of RNA in living cells (about 80% of total RNA in a cell)
• Serves as a scaffold of the ribosomal subunits
• Ribosomal RNA associates with proteins to form ribosomes (the site of protein synthesis).
• Catalyzes specific chemical reactions (ribozymes: “ribo-” that acts like en-“zymes”)
• Largest rRNA 23S (prokaryotes) and 28S (eukaryotes) in the large subunit of the ribosome:
  • The most important ribozyme
  • Ribosomal RNA is a peptidyl transferase (it catalyzes peptide bond formation between amino acids to form proteins).
• Note the differences:
  • Ribosomal RNA: rRNA
  • Ribosome: rRNA + proteins
  • Ribozyme: rRNA with enzyme-like activity, catalyzing protein synthesis in ribosomes

Other Forms of RNA

RNA processing

Small nuclear RNA:

• Non-coding RNA (eukaryotes) 
• Associates with proteins to form small nuclear ribonucleoproteins (snRNPs):
  • Small nuclear ribonucleoproteins (snRNPs) + protein = spliceosome
  • Spliceosomes excise introns from a transcribed pre-mRNA (splicing).
  • Key for rRNA and mRNA processing and gene regulation

Small nucleolar RNA:

• Non-coding RNA (eukaryotes)
• Modifies RNA nucleotides

Gene regulation

Micro RNA:

• Non-coding RNA (eukaryotes)
• Regulates degradation and translation of mRNA
• Abnormal expression of miRNA can contribute to malignancy development (functioning as oncogenes or tumor suppressors).

Small regulatory RNA:

• Non-coding RNA (prokaryotes)
• Gene regulation

6sRNA:

• Transcription regulation

Genome defense

Small interfering RNA:

• Non-coding RNA (eukaryotes)
• Silencing RNA
• Operates within the RNA interference (RNAi) pathway (with miRNA)
• Defense against foreign RNA

PIWI-interacting RNA:

• Non-coding RNA (eukaryotes)
• Regulates gene expression and fights viral infection

CRISPR (crRNA):

• Non-coding RNA (prokaryotes)
• Defense against foreign DNA and RNA

Other ribozymes

Ribonuclease P (RNAse P):

• Cleaves pre-tRNA to generate the 5’ free end of mature tRNA

Self-splicing introns:

• Introns in the genes act as ribozymes with nuclease activity.

Viroid:

• Non-coding RNA
• Naked RNA in plants
• Cleaves itself during the viroid replicative cycle