Post-transcriptional Modifications (RNA Processing)

Post-transcriptional modifications (PTMs) are processes that facilitate the generation of mature, functional RNA. These rapidly responsive regulatory mechanisms allow different proteins to be produced from one gene and act as regulators of the phenotype and proliferation rate. These modifications also play a role in some forms of cancer and neurodegenerative diseases. The pre-messenger RNA (mRNA), called heterogeneous nuclear RNA (hnRNA), is modified by adding a 5’ 7-methylguanosine cap and a 3’ poly-A (polyadenylate) tail for stability and protection. Moreover, hnRNA that contains introns (noncoding sequences) among the expressed sequences or exons undergo splicing. This process removes introns to produce a mature mRNA carrying the coding sequence for translation. Alternative splicing, on the other hand, also excludes the introns, but varying combinations of exons are linked, producing different proteins from the original mRNA. In RNA editing, the mRNA sequence is altered and differs from the transcribed DNA template. Transfer RNA and ribosomal RNA start from longer precursor molecules and go through steps that include methylation, trimming, and addition of nucleotides.

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Overview

Transcription

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.
Gene expression from dna

Gene expression from DNA, the genetic sequence, is transcribed into the RNA (transcription):
Transcription of genetic information is the first step in gene expression and is the process through which a coding region of DNA (double-stranded structure) is used as a template for the synthesis of messenger RNA (mRNA). The mature mRNA is translated into amino acids, forming proteins (translation) with the help of ribosomal RNA and transfer RNA (tRNA). This image shows transcription without post-transcriptional modifications of the RNA.

Image by Lecturio.

Modifications

Primary transcripts, or immediate products of transcription, undergo alterations to become biologically functional.

  • mRNA:
    • 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
      • Splicing
    • 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.
Summary of post-transcriptional modifications of hnrna

Summary of post-transcriptional modifications of hnRNA into a mature mRNA:
The addition of the 5’ cap and the 3’ poly-A tail and splicing (removal of the intervening sequences or introns)

Image by Lecturio.

Addition of the 5′ Cap and 3′ Poly-A Tail

5′ cap

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))

Functions:

  • 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

Function:

  • Prevents degradation in the cytosol by 3′ exoribonucleases
  • Stabilizes mRNA
Post-transcriptional modifications of rna

Post-transcriptional modifications of RNA:
The 5’ cap (7-methylguanosine) and 3’ poly-A tail modifications prevent degradation of the mRNA in the cytosol.

Image by Lecturio.

Heterogeneous Nuclear RNA Splicing

Exons and introns

Heterogeneous nuclear (pre-mRNA) contains:

  • Coding sections called exons (expressed sequences)
  • Noncoding sections called introns (intervening sequences)

Processing:

  • hnRNA needs processing (splicing) to produce the mRNA carrying the proper coding sequences.
  • Occurs in most eukaryotic genes, most commonly on mRNA
Pre-mrna exons and introns with an overview of splicing

Pre-mRNA exons and introns with an overview of splicing (from top to bottom):
Pre-mRNA transcript contains exons and introns. Interactions of the transcript with small nuclear ribonucleoproteins and other proteins form a spliceosome at certain junctions of the transcript. Cuts are made at the splice sites, and the intron is released. Spliced RNA now only has exons, which contain the coding sequence.

Image by Lecturio.

Splicing

  • 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
  • Mechanism:
    1. Small nuclear ribonucleoproteins recognize the splice sites and branch site owing to the base sequences on the hnRNA.
    2. hnRNA, small nuclear ribonucleoproteins, and other proteins combine to form the spliceosome. 
    3. 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).
    4. The now free 5’ terminus of the intron links to the branch site, forming a loop, or lariat, structure.
    5. 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:
    • β-Thalassemia: 
      • 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).
Technical aspects of splicing

Technical aspects of splicing:

The pre-mRNA/hnRNA are made up of exons and introns. Small nuclear ribonucleoproteins + other proteins recognize the branch site and the exon–intron junctions where to cut: the 5’ donor site (containing the invariant GU sequence) and the 3’ acceptor site (containing the invariant AG sequence). The transcript of hnRNA + small nuclear ribonucleoproteins + other proteins combine at these sites and form the spliceosome.
Top image: Through the aid of small nuclear ribonucleoproteins (snRNPs), the first cut is made by the adenylyl residue (in the branch site) via a nucleophilic attack on the 5’ donor site.
Middle image: The free 5’ terminus then forms a bond with the branch site (making the lariat structure).
Bottom image: The second cut is made on the 3’ site of the intron and the exons are joined.

Image by Lecturio.

Alternative splicing

  • Differential splicing of one hnRNA sequence
  • Mechanisms:
    • 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)
Examples of alternative splicing

Examples of alternative splicing:
Protein A: Exons 1–5 were joined after splicing of introns.
Proteins B and C: An exon was selectively excluded to form a different protein.

Image by Lecturio.

RNA Editing

Generally, the DNA sequence is reflected in the mature mRNA. Alteration of the sequence or RNA editing is an exception.

Definition

  • 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
  • Processes:
    • Base changes
    • Base deletions
    • Base insertions

Base changes

“C-to-U” editing:

  • 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).

“A-to-I” editing:

  • 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

Insertions/deletions

  • Occur in trypanosomes and related protozoa, affecting the mitochondrial mRNA
  • Involves addition or deletion of uridine

Ribosomal RNA and Transfer RNA Processing

Ribosomal RNAs

  • 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 RNAs

  • 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
    • Introns
  • 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
Transfer rnas (trna)

Secondary structure of transfer RNA (tRNA). Note that its entire sequence can be seen, indicating the reduced size.

Image by Lecturio.

Related videos

References

  1. 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
  2. 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.
  3. 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
  4. 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

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