Post-Transcriptional Modification

The mRNA transcript synthesized by RNA polymerase II is modified

in eukaryotes.

Steps of mRNA Processing

• 5’ Capping

• 3’ polyadenylation

• Intron Removal and Exon Splicing

Nascent RNA transcripts from protein-coding genes are called pre-mRNA .They are associated with proteins until they are exported into the cytosol. These proteins and the mRNA are collectively called heterogenous ribonucleoprotein particles (hnRNP).

Functions of hnRNP

• Prevent folding of pre-mRNA into secondary structures that may inhibit its interactions with other proteins.

• May associate with the splicing apparatus.

• Transport of mRNA out of the nucleus.

5’ CAPPING

After the mRNA transcript is about 25-30 nucleotides long a 7-methylguanosine is added to the 5’ end.

The capping enzyme associates with the phosphorylated CTD of RNA polymerase II.

Protects the 5’ from enzymatic degradation in the nucleus and assists in export to the cytosol.

3’POLYADENYLATION

• Primary transcript is cleaved at a poly A tails is added to the free 3’ OH.

• Up to 250 A residues may be added.

• Carried out by poly(A) polymerase

• Protects the mRNA from degradation from the 5’ end.

RNA SPLICING

• Introns are removed and the exons are spliced together.

• Splice sites occur at both the 5’ and 3’ ends of introns.

• The intron is cut at the splice sites.

• Only 30-40 nucleotides at each end of an intron are required for splicing.

Splicing Mechanism – PART 1

• Five U-rich small nuclear RNAs (snRNA) participate in splicing.

• These are designated U1, U2, U4, U5 and U6.

• The snRNAs are each associated with 6-10 proteins in small nuclear ribonucleoprotein particles (snRNPs).

SPLICING MECHANISM – PART 2

• 5’ end of U1 base pairs with the 5’ splice site of the mRNA.

• U2 pairs with the branch point A. The branch point A is an invariant nucleotide present 20-50 bases upstream from the 3’ splice site in an intron.

• The other snRNPs bind to the pre-mRNA forming a spliceosome.

SPLICING MECHANISM – PART 3

• U1 and U4 are released.• Two transesterification reactions remove the

intron (now called a lariat intron).• The second transesterification reaction ligates

the exons.• The lariat intron is converted into a linear RNA.• Nuclear exonucleases cut the linear RNA from

both the 3’ and 5’ ends, thus recycling the nucleotides.

Trans-Splicing

• Functional mRNAs (mature mRNAs) are derived from a single pre-mRNA in most eukaryotes.

• However, in some, a mature mRNA may be derived from separate RNA molecules (for example in trypanosomes [which cause sleeping sickness] and euglenoids).

• This process is called trans-splicing.

Ribozymes

• These are catalytic RNA molecules.

• Include:– 5S and 23S rRNA of ribosomes that has

peptidyl transferase activity.– Group I self-splicing introns of nuclear DNA of

protozoans.– Group II self-splicing introns of mitochondrial

and chloroplast DNA.

Alternative Splicing

• Synthesis of a mature MRNA from different exons from the same gene in different cells.

• Resultant proteins are called isozymes.

• Example: Slo isoforms in the hair cells of the cochlea (inner ear) of vertebrates. Slo is a gene that encodes a K+ channel that opens in response to changing [Ca+2].

RNA Editing

• Rare phenomenon that occurs most often in the mitochondria of plants and protozoans, as well as in chloroplasts.

• Nucleotides in exons are changed prior to the production of the mature mRNA.

Transport Across the Nuclear Membrane

• Molecules of all sizes enter and leave the nucleus via Nuclear Pore Complexes (NPC).

• Consist of multiple copies of different proteins called nucleoporins. From 50-100 different proteins (depending on organism) are involved.

• Complex is extremely large, about 30 times larger than a ribosome.

Nuclear Pore Complex

Molecular trafficking between the nucleus and the cytoplasm occurs via nuclear pore complexes (NPCs). The NPCs allow passive diffusion of ions and small molecules. Nuclear proteins, RNAs, and ribonucleoprotein (RNP) particles larger than ~9 nm are selectively and actively transported through NPCs by a signal-mediated and energy-dependent mechanism.

Import into the Nucleus

• Proteins synthesized in the cytosol and meant for use in the nucleus enter via the NPC.

• Such proteins contain a nuclear-localization signal (NLS) that directs their translocation into the nucleus.

• The signal is a short sequence of amino acids near the C-terminal end.

•

Summary of Import/Export of Proteins

through the NPC.

MRNA Export through the NPC

Mediated by an mRNA-exporter