Lecture 17/18 (RNA Transription)

5’ Cap Addition

• modified guanine structure

• added to the front of the 5’ end of mRNA’s

• linked 5’-5’ triphosphate bond

• occurs after 20-30 base pairs

5’ Cap Function

• transport: interaction with cap binding complex ensures proper export to the cytosol from the nucleus

• protection: prevents 5’-3’ RNA exonuclease digestion

• activity: serves as a docking site for translational/machinery ribosome

3’ End Formation of eukaryotic mRNA (poly-A tail)

• poly(A) signal (PAS), right next to cleavage site, which is between PAS and Downstream Signal Element (DSE)

• Cleavage site is cut

• Poly-A tail is added next to PAS, which now has Cleavage and Polyadenylation Specific Factor (CPSF)

RNA Splicing

• introns are removed, exons are joined

• needed for typical eukaryotic mRNA before it can be used to produce a correct protein through translation

• spliceosome

• (on average, eukaryotic gene contains about 4bp of protein coding region)

Size of introns/exons

• Commonly, introns are 40-150 nt long

• minimum size is ~20 nt (dictated by the ability of the splicing machinery to assemble on the intron

• in contrast, exons are generally short; narrow size distribution of 50-300 nt

Spliceosome

• highly dynamic structure; constantly rearranges during a pre-mRNA splicing cycle

• components: specific pre-formed RNA-protein complexes that bind together with other proteins to different regions of the introns

  • RNA-protein particles:

    1. non-coding RNA, U snRNA’s

    2. U snRNA-specific proteins (ribonucleoproteins - U snRNPs)

Modes of Alternative Splicing

• Exon skipping/inclusion

• Alternative 3’ splice sites

• Alternative 5’ splice sites

• Mutually exclusive exons

• Intron retention

Alternative Splicing

Different combinations of exons could be spliced together to produce different mRNA isoforms of a gene.

Considered one of the main mechanisms responsible for increasing the diversity and complexity of the human genome

A gene in the genome

• 5’ Cap

• 5’ untranslated region (5’UTR) - varies in length, follows 5’ Cap

• coding sequence (open reading frame - ORF) - specific amino acid sequence of the protein that will be produced during translation; varies in length according to size of the protein

• 3’ UTR - varies in length and contains information influencing the stability of the mRNA

• poly-A tail follows 3’ UTR

• translation starts after 5’ UTR and stops before 3’ UTR

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