Genetic information from DNA is copied into messenger RNA (mRNA).
- In this process, mRNA is synthesized from the 5’ end to the 3’ end.
- The initial transcript is known as heterogeneous nuclear RNA (hnRNA) or pre-mRNA.
Primary transcripts, or immediate products of transcription, undergo alterations to become biologically functional.
- Prokaryotes: Most primary mRNAs have no modifications.
- Eukaryotes: Synthesized transcript of mRNA (or hnRNA) undergoes processing before leaving the nucleus.
- Addition of 5’ cap
- Addition of 3’ poly-A tail
- Modification of hnRNA produces mature mRNA, which is transported to the cytoplasm through nuclear pores.
- In some cases, RNA editing occurs with base changes, creating a sequence different from that copied from the DNA.
- A different mRNA sequence produces a different protein; this varies from the old hypothesis of “one gene–one polypeptide.”
- Transfer RNA (tRNA) and ribosomal RNA (rRNA):
- Structural molecules that are not translated
- Both have pre-tRNAs and pre-rRNAs that undergo processing.
Addition of the 5′ Cap and 3′ Poly-A Tail
7-Methylguanosine (methylated guanylyl residue) is added to the 5’ end of hnRNA via:
- Removal of the leading phosphate group at the 5’ terminal by RNA triphosphatase
- Transfer of guanosine monophosphate (GMP) from the guanosine triphosphate group by guanylyl transferase
- Methylation of guanine by guanine-7-methyltransferase (methyl group from S-adenosylmethionine (SAM))
- Prevents exonuclease degradation
- Recognition sequence for translation
3′ Poly-A tail
50 to 250 adenylyl residues (AMP) are added to the 3’ end of hnRNA via:
- Cleaving of about 20 nucleotides downstream from an AAUAA recognition sequence
- Addition (and extension up to 250 nucleotides) of poly-A tail (generated from ATP) by poly-A polymerase
- Prevents degradation in the cytosol by 3′ exoribonucleases
- Stabilizes mRNA
Heterogeneous Nuclear RNA Splicing
Exons and introns
Heterogeneous nuclear (pre-mRNA) contains:
- Coding sections called exons (expressed sequences)
- Noncoding sections called introns (intervening sequences)
- hnRNA needs processing (splicing) to produce the mRNA carrying the proper coding sequences.
- Occurs in most eukaryotic genes, most commonly on mRNA
- Removal of introns from the hnRNA/pre-mRNA, while linking the exons to form the mature mRNA
- Process involves the hnRNA and additional components:
- Small nuclear ribonucleoproteins (which are made up of small nuclear RNAs (snRNAs) and proteins)
- Other binding proteins
- Junctions where splicing reaction occurs:
- Splice sites:
- Areas where cuts are made between the exon and intron
- Base sequences identify these sites, one at the 5’ side (beginning of the intron) and the other at the 3’ side (end of the intron).
- 5’ site/donor splice site: invariant GU
- 3’ site/acceptor splice site: invariant AG
- Branch site: located upstream from 3’ site
- Splice sites:
- Small nuclear ribonucleoproteins recognize the splice sites and branch site owing to the base sequences on the hnRNA.
- hnRNA, small nuclear ribonucleoproteins, and other proteins combine to form the spliceosome.
- The spliceosome complex makes a cut on the 5’ donor splice site (occurs via a nucleophilic attack by an adenylyl residue in the branch site).
- The now free 5’ terminus of the intron links to the branch site, forming a loop, or lariat, structure.
- The 3’ splice site is recognized, and the second cut occurs there. Release of the lariat follows, and the 2 exons are joined to form the mature RNA
- Occurs simultaneously with the 5’ cap and 3’ poly-A tail hnRNA modifications
- Related conditions:
- Contains defects in mRNA splicing of β-globin gene
- Significant homozygous mutations (thalassemia major) result in transfusion-dependent anemia
- Spinal muscle atrophy:
- Lack of functioning SMN1 gene due to mutation
- The remaining SMN2 is unable to compensate because of the defect at the level of pre-mRNA splicing (skipping of exon 7).
- Differential splicing of one hnRNA sequence
- Exons are selectively included or excluded.
- Alternative 5′ donor or 3′ acceptor sites are used.
- Polyadenylation sites can differ.
- Up to 95% of multi-exon genes undergo alternative splicing (AS) to encode proteins with different cellular functions.
- AS is a rapidly responsive regulation step needed for fine-tuning protein synthesis and thereby determining cell phenotypes and proliferation rates.
- Approximately 15% of hereditary diseases and cancers are reported to be associated with AS.
- Different combinations of exons can lead to different related proteins being created from the same hnRNA:
- Immunoglobulin molecules (genes for heavy chains have exons related to individual subtypes)
- Tropomyosin variants in muscle
- Dopamine receptors in the brain (D2 receptors with 2 isoforms)
Generally, the DNA sequence is reflected in the mature mRNA. Alteration of the sequence or RNA editing is an exception.
- A change in the coding information after transcription results in the sequence of RNA differing from that of its original DNA.
- Believed to contribute to genetic regulation
- Base changes
- Base deletions
- Base insertions
- Apolipoprotein B (apoB) gene: The same gene codes for ApoB100 (synthesized in the liver) and ApoB48 (synthesized in the intestine).
- In the intestine:
- Deamination of cytosine (to uracil) catalyzed by the enzyme cytidine deaminase.
- CAA (cytidine–adenine–adenine) codon (in the mRNA) → UAA (uridine–adenine–adenine), a termination signal or a stop codon
- As a result of this editing:
- ApoB100 is a 100-kDa protein made of 4536 amino acids.
- ApoB48 is a shortened 48-kDa protein made of 2152 amino acids.
- The change produces ApoB48, which lacks the C-terminal domain of ApoB100 (responsible for LDL receptor binding).
- Affects double-stranded RNA substrates
- Deamination of adenine (to hypoxanthine) catalyzed by ADARs (adenosine deaminase acting on RNA)
- Adenosines converted into inosines, which leads to A-to-G base substitutions
- Generates alternative splice sites
- Occur in trypanosomes and related protozoa, affecting the mitochondrial mRNA
- Involves addition or deletion of uridine
Ribosomal RNA and Transfer RNA Processing
- In prokaryotes:
- As studied in Escherichia coli, 3 types of rRNAs (5S, 16S, and 23S) have polycistronic primary transcripts.
- Initial processing of transcript via endonucleolytic cleavage by RNases → pre-rRNA
- The 5’ and 3’ ends of pre-rRNA are then trimmed by another set of RNases.
- Methylation occurs during assembly of ribosomes to protect from degradation.
- In eukaryotes, primary transcript is a large 45S precursor molecule:
- Contains rRNA molecules (28S, 18S, and 5.8S) with spacer sequences in between
- Processed in the nucleolus:
- By methylation at numerous sites (facilitated by small nucleolar RNAs (snoRNAs))
- Then by cleaving and trimming of the RNA
- Fourth type of rRNA, 5S, is processed separately.
- The resultant mature rRNAs, which associate with other proteins, become the scaffold of the ribosomal units in the cytoplasm.
- Some eukaryotic rRNA have introns, and in those, pre-rRNAs are self-splicing (act as ribozymes).
- Transfer RNA is a single polynucleotide made up of an average of 75 nucleotides with unique characteristics:
- Due to distinct folding, the 5’ end and 3’ end make up the acceptor stem.
- The 3’ end also has a CCA (cytosine–cytosine–adenine) sequence.
- Has modified bases such as inosine, dihydrouridine, and pseudouridine
- The precursor tRNA contains:
- Extra nucleotides at the 3’ and 5’ ends
- Processing involves:
- Removal of the extra nucleotides and introns
- Modification of standard bases (such as methylation and deamination)
- Utilizes RNase P (a ribozyme) to form the 5’ end
- Addition of CCA at the 3’ end by tRNA nucleotidyltransferase
- Helm M. (2006). Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Research 34(2):721–733. https://doi.org/10.1093/nar/gkj471
- Weil P. (2018). RNA synthesis, processing, & modification. Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil P (Eds.), Harper’s Illustrated Biochemistry, 31st ed. New York: McGraw-Hill.
- Jiang W, Chen L. (2020). Alternative splicing: human disease and quantitative analysis from high-throughput sequencing. Computational and Structural Biotechnology Journal 19:183–195. https://doi.org/10.1016/j.csbj.2020.12.009
- Anna A, Monika G. (2018). Splicing mutations in human genetic disorders: examples, detection, and confirmation. Journal of Applied Genetics 59(3):253–268. https://doi.org/10.1007/s13353-018-0444-7