RNA processing is a crucial step in gene expression. It involves three main steps: capping, splicing, and polyadenylation. These modifications protect the mRNA, help it leave the nucleus, and make it ready for translation.

Capping adds a special structure to the start of the mRNA. Splicing removes unnecessary parts and can create different versions of proteins. Polyadenylation adds a tail to the end of the mRNA. Together, these steps ensure mRNAs are stable and can be efficiently translated into proteins.

RNA Processing: Capping, Splicing, and Polyadenylation

Process of 5' capping

• Occurs co-transcriptionally after synthesis of first 20-30 nucleotides of nascent pre-mRNA
  • RNA triphosphatase removes gamma phosphate from 5' end of nascent pre-mRNA
  • Guanylyltransferase catalyzes addition of GMP to 5' end of pre-mRNA forming 5'-5' triphosphate linkage
  • Methyltransferase methylates N7 position of guanine cap producing mature 7-methylguanosine (m7G) cap
• 5' cap plays crucial roles in mRNA stability and translation
  • Protects mRNA from 5' to 3' exonucleolytic degradation (exosome complex)
  • Facilitates export of mature mRNA from nucleus to cytoplasm (nuclear pore complex)
  • Enhances translation efficiency by promoting recruitment of translation initiation factors (eIF4E) and 40S ribosomal subunit

Mechanism of RNA splicing

• Removes introns and joins exons to form mature mRNA
• Carried out by spliceosome, a large ribonucleoprotein complex composed of five small nuclear ribonucleoproteins (snRNPs) and numerous associated proteins (U1, U2, U4, U5, U6)
• Splicing reaction occurs in two transesterification steps:
  1. 2' hydroxyl group of branch point adenosine within intron attacks 5' splice site forming lariat structure
  2. 3' hydroxyl group of upstream exon attacks 3' splice site joining exons and releasing lariat intron
• Allows for generation of diverse protein isoforms from single gene through alternative splicing (CD44, tropomyosin)
  • Different combinations of exons can be included or excluded in mature mRNA
  • Expands coding potential of genome and contributes to proteome diversity

Constitutive vs alternative splicing

• Constitutive splicing: all exons in pre-mRNA are included in mature mRNA
  • Constitutively spliced exons always present in final mRNA product (β-actin)
  • Ensures production of consistent protein product from gene
• Alternative splicing: selective inclusion or exclusion of specific exons in mature mRNA
  • Exons can be skipped, mutually exclusive, or have alternative 5' or 3' splice sites (FGFR2, Bcl-x)
  • Allows for production of multiple protein isoforms from single gene
  • Regulated by cis-acting regulatory elements (exonic/intronic splicing enhancers/silencers) and trans-acting factors (SR proteins, hnRNPs)
• Both play essential roles in regulating gene expression and protein diversity

Significance of polyadenylation

• Addition of poly(A) tail to 3' end of mature mRNA
• Polyadenylation process involves:
  • Cleavage of pre-mRNA at polyadenylation signal (AAUAAA) by cleavage and polyadenylation specificity factor (CPSF)
  • Addition of poly(A) tail (200-250 adenosine residues) by poly(A) polymerase (PAP)
• Poly(A) tail plays important roles in mRNA stability and translation efficiency
  • Protects mRNA from 3' to 5' exonucleolytic degradation (exosome complex)
  • Facilitates export of mature mRNA from nucleus to cytoplasm (nuclear pore complex)
  • Enhances translation efficiency by promoting formation of closed-loop structure through interactions between poly(A) binding protein (PABP), eIF4G, and 5' cap
• Regulation of poly(A) tail length can influence mRNA stability and translation
  • Deadenylation (shortening of poly(A) tail) often first step in mRNA decay (CCR4-NOT complex)
  • Cytoplasmic polyadenylation can activate translation of dormant mRNAs in specific cellular contexts (oocyte maturation, synaptic plasticity)