Chapter 8:Post-Transcriptional gene Controls and Nuclear

Transport

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Posttranscriptional gene control:

The Molecular mechanism after the initiation of transcription

Pre-mRNA processing (3 steps) in Pre-mRNA processing (3 steps) in eukaryoteseukaryotes

5’cap is added to nascent RNAs shortly after 5’cap is added to nascent RNAs shortly after initiation by RNA polymerase IIinitiation by RNA polymerase IICapping Enzyme mediated process (only for mRNA)Capping Enzyme mediated process (only for mRNA)

Why capping????Why capping????

Second step: Second step: polyadenylation of pre-polyadenylation of pre-mRNAmRNA

Note:Note:Two poly A signals: sites for the Two poly A signals: sites for the binding of cleavage factorsbinding of cleavage factors

Poly A tail/protein Complex: provide a Poly A tail/protein Complex: provide a site for PAP (Poly A Polymerase) site for PAP (Poly A Polymerase)

Dual roles of PAP: cut G/U site away Dual roles of PAP: cut G/U site away and add poly Asand add poly As

How many As are added and potential How many As are added and potential role?: 200As and protect mRNA from role?: 200As and protect mRNA from endonucleases.endonucleases.

http://vcell.ndsu.nodak.edu/animations/

G/U: G/U or U rich region

CPSF: cleavage and polyadenylation specificity factor

CStF: cleavage stimulatory factor

CFI: cleavage factor I

CFII: cleavage factor II

STEP 3:STEP 3:Alternative splicing (alternative Alternative splicing (alternative combination of exons): a common combination of exons): a common mechanism to produce different mechanism to produce different proteins from one transcription unit.proteins from one transcription unit.

Ex. Fibronectin isoforms Ex. Fibronectin isoforms

Classic experiment (1977)Classic experiment (1977): : Early evidence that introns Early evidence that introns removed during splicing came from electron microscopy of DNA-removed during splicing came from electron microscopy of DNA-mRNA hybridsmRNA hybrids

SPLICING MECHANISM SPLICING MECHANISM Consensus sequence around 5’ and 3’ splice Consensus sequence around 5’ and 3’ splice

sites in vertebrate pre-mRNAsites in vertebrate pre-mRNA

NOTE:

5’ and 3’ invariant sequences in Introns?

One more invariant sequence in Introns?

What is meant by pyrimidine? (CU)

SPLICING MECHANISM :

How is an intron removed?

Two transesterification reactions that result in removal of an intron in pre-mRNA.

SPLICING MECHANISMSPLICING MECHANISM

At least five snRNPs (At least five snRNPs (Small nuclear Ribonuclear Small nuclear Ribonuclear proteins, also called U proteinsproteins, also called U proteins) associate to form ) associate to form spliceosome to splice pre-mRNA spliceosome to splice pre-mRNA - snRNP (U): - snRNP (U): Named after?Named after? (Uracil) (Uracil)

NoteNote- How are snRNPs recruited?- How are snRNPs recruited?

- Why so complex?- Why so complex?

- Two transesterification- Two transesterification

- Final products?- Final products?

- Destination of intron?- Destination of intron?

SPLICING MECHANISMSPLICING MECHANISM: RNA WORLD.: RNA WORLD.SnRNPs contain U-rich RNA sequences that bind the intron, which will be SnRNPs contain U-rich RNA sequences that bind the intron, which will be taken awaytaken away

NoteNote: : Complementary base pairing.Complementary base pairing.The consequence of point mutation and compensation mutationThe consequence of point mutation and compensation mutation

http://vcell.ndsu.nodak.edu/animations/

Finished Splicing, but one more modification before exit through nuclear pore

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RNA EditingRNA Editing: A single base change in pre-mRNA, called : A single base change in pre-mRNA, called RNA editingRNA editing, results in , results in production of a functionally different protein. A final point is that the sequence that production of a functionally different protein. A final point is that the sequence that is found in the DNA is not entirely and faithfully represented in the final protein is found in the DNA is not entirely and faithfully represented in the final protein product.product.

Editing process: is it error? Or Planned?

What mediates this editing process?

What is the consequences of editing?

Why RNA editing?

it is an efficient way to create proteins with slightly different functions to use in specialized circumstances.

Mature mRNA made via 3 or 4 Mature mRNA made via 3 or 4 steps of post-transcription steps of post-transcription

modificationmodification

Next rounds of post-transcriptional control

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Macromolecular transport across the nuclear envelope.Macromolecular transport across the nuclear envelope.Nuclear pore complex (NPC) is composed of 30 different proteinsNuclear pore complex (NPC) is composed of 30 different proteinsIntegral proteins (nucleoporins)Integral proteins (nucleoporins)High FG (Phe, Gly) contents (FG-nucleoporins)High FG (Phe, Gly) contents (FG-nucleoporins)Establish a molecular sieve (Free pass for molecules < 40Kd)Establish a molecular sieve (Free pass for molecules < 40Kd)A plug (transporter) is associated with FG-nucleoporins to block the passage of A plug (transporter) is associated with FG-nucleoporins to block the passage of bigger moleculesbigger molecules

http://www.youtube.com/watch?v=dJLeLRXIOj0&NR=1

Mature mRNA structure just before the translocationNotice many mRNA binding proteins (mRNP) and mRNA transporter proteins (mRNA exporter) are associated with the mRNA

Export mRNA through NPCImportant Events

- Interaction of dimeric mRNP with mRNA and mRNA exporter

- Hydrophobic interaction of mRNA exporter with FG repeats of NPC proteins

- mRNA exporter translocates mRNP-mRNA complex

Posttranscriptional control viaRegulation of translation initiation

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Translation:http://vcell.ndsu.edu/animations/translation/index.htm

A very important Post-A very important Post-transcriptional controltranscriptional control via via small interference RNA (small interference RNA (to to

regulate translation process): in regulate translation process): in cytoplasm.cytoplasm.

Consequence?

1000 of DsRNA expressed in specific cell types at particular times

Si RNA (how many bases?)

Dicer (endonuclease)

RISC (RNA-Induced Silencing complex)

-interacts and unwinding

-ribonuclease activityhttp://www.hhmi.org/biointeractive/rna/rnai/index.html

Post-transcription control via degradation of mRNAPost-transcription control via degradation of mRNAmRNAs are degraded by several mechanisms in the mRNAs are degraded by several mechanisms in the cytoplasm.cytoplasm.

Consequence?Consequence?

Degradation mediated byDegradation mediated by- decapping enzyme- decapping enzyme- deadenylase- deadenylase- endonuclease- endonuclease

Processing of rRNA in nucleolusProcessing of rRNA in nucleolusPre-rRNA (45 S) from nucleolus of a frog oocyte

I: pre-rRNAII: mRNAIII: tRNAs, 5S rRNA, small stable RNAs

Processing of tRNA

Pre-tRNA precursor is made by RNA polymerase III. Processing of tRNA includes trimming 5’ sequence, replacing 3’UU with CCA, and modifying multiple internal bases

Overview of RNA processing and posttranscriptional gene control