alternative splicing controls sex determination in drosophila

McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010

© R. Roy, 2010© R. Roy, 2010

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© R. Roy, 2010© R. Roy, 2010

Alternative splicing controls sex determination in Drosophila

Sexually dimorphic characteristics


© R. Roy, 2010© R. Roy, 2010

Alternative splicing controls sex determination in Drosophila


© R. Roy, 2010© R. Roy, 2010

Polyadenylation of the 3’ end of the nascent transcript is required for

stability• Polyadenylation occurs in

two phases:

A slow phase mediated by Poly A Polymerase (PAP) during which approximately 12 A residues are added on to the cleaved 3’ end.

This structure is recognised by Poly A Binding Protein II (PABPII-to distinguish it from the cytoplasmic PABP), which catalyses the rapid addition of ~200 A residues.


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Splicing variants or isoforms provide added diversity: Alternative Splicing


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Protein synthesis IProtein synthesis I

chapter 4chapter 4


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How Is the Information Content in How Is the Information Content in DNA Transcribed to Produce RNA?DNA Transcribed to Produce RNA?

The genetic code: specifies which amino acids will be used to build a protein

Codon: a sequence of three bases. Each codon specifies a particular amino acid.

Start codon: AUG—initiation signal for translation (translational start site)

Stop codons: stops translation and polypeptide is released (translational termination signal)


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The three roles of RNA in The three roles of RNA in protein synthesisprotein synthesis


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Figure 12.6 The Genetic Code


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Figure 12.8 Transfer RNA

anticodon


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Pre-tRNAs are transcribed by RNA Pol III and thereafter are processed to yield mature tRNA

• 1) the 5’ end sequence is removed

• 2) a short intron is removed

• 3) CCA is added on to the 3’ end

• 4) extensive modification of internal bases

Pre-tRNAs undergo critical processingPre-tRNAs undergo critical processing


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Pre-rRNA transcription units are Pre-rRNA transcription units are arranged in repetitive clustersarranged in repetitive clusters

Since rRNA accounts for approximately 80% of the total RNA in the cell, it has to be transcribed extensively to be able to satisfy the continuous demand


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Eukaryotic rRNA is transcribed in Eukaryotic rRNA is transcribed in units comprising the 18S, 5.8S and units comprising the 18S, 5.8S and

28S rRNAs28S rRNAs

the arrangement 5'-3' 18S - 5.8S - 28S is invariant within and between species


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the pre-rRNA transcript is extensively processed in the nucleolus. Not only is it cleaved into its components, but also numerous bases are chemically modified.

all the pre-rRNA processing is executed through small nucleolar RNAs, which are part of snoRNPs.

unlike the other rRNAs encoded by the single pre-rRNA molecule, 5S rRNA is transcribed outside the nucleolus and arrives there by diffusion and is not further processed

Precursor rRNA is extensively Precursor rRNA is extensively processedprocessed


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rRNA folds into conserved stem-rRNA folds into conserved stem-loop structuresloop structures

the overall stem-loop structure of ribosomal RNA is highly conserved among the thousands of species investigated to date.

(molecule shown is 16S bacterial rRNA, similar to 18S eukaryotic rRNA)


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rRNA is an integral part of the rRNA is an integral part of the protein synthesizing machine, the protein synthesizing machine, the

ribosomeribosome


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Assembly of the pre-initiation complex


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Protein synthesis can be negatively regulated by phosphorylation of eIF2.


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RibosomesRibosomes


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The tRNAThe tRNAiiMetMet is needed to start is needed to start

synthesis of a polypeptide chainsynthesis of a polypeptide chaintwo types of tRNAs

exist for the methionine codon

both are charged by

the same aminoacyl

tRNA synthetase

tRNAMet is exclusively used for elongation of a polypeptide chain

tRNAiMet is exclusively

used for initiation of a polypeptide chain

the methionine in bacterial tRNAi

is modified by addition of a formyl group


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A 5' cap is required for the efficient A 5' cap is required for the efficient translational initiation of eukaryotic translational initiation of eukaryotic

mRNAsmRNAs Because the dimeric

guanyltransferase binds to the phosphorylated C-terminal domain (CTD) of RNA polymerase II only mRNA transcripts are capped!

therefore, only class II (mRNA) transcripts are efficiently translated!


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• Sonenberg et al. purified a protein that bound the 7mGDP cap specifically using an affinity chromatography approach

• Addition of the purified protein to capped mRNAs increased the translational efficiency in vitro, but did not affect uncapped transcripts.

A cap-binding protein is A cap-binding protein is required for efficient required for efficient

translational initiationtranslational initiation

X


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eIF4 is instrumental in recruiting eIF4 is instrumental in recruiting an mRNA to the preinitiation an mRNA to the preinitiation

complexcomplex next, a protein

complex named eIF4 binds the pre-initiation complex as well as the cap of an mRNA.

An associated helicase activity removes any secondary structure in the 5’ region of the transcript...


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The initiation complex uses ATP energy and the helicase activity of eIF4A to scan the mRNA until the

initiation codon is found (usually the first AUG).

eIF4E-CapeIF4G-binds eIF3eIF4A-RNA helicase (ATP)

ACC G


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eIF4 and mRNA are recruited to form an initiation complex

Activity of the cap-binding subunit eIF4E can also be regulated.


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Bacterial translation initiation Bacterial translation initiation requires the Shine-Dalgarno boxrequires the Shine-Dalgarno box

the ribosome has to recognize the start codon of the ORF the ribosome start site for protein synthesis is directed toward the AUG

initiator codon by the Shine-Dalgarno box, a 5-8bp sequence that can base pair with the 16S rRNA molecule in the small ribosomal subunit


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The Shine-Dalgarno box positions the The Shine-Dalgarno box positions the AUG initiation codon for pairing with AUG initiation codon for pairing with

the tRNAthe tRNAii the pairing of the Shine-Dalgarno box with the 16S rRNA results in a match of the initiation AUG codon with the initiator tRNA (tRNAi)

in mammalian cells a relatively conserved

sequence ACCAUGG recognises the start site (AUG)- KOZAK sequence (after Marilyn KOZAK)


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Recruitment of the large ribosomal subunit completes the

initiation phase.

Commitment to translation(can no longer dissociate)

alternative splicing controls sex determination in drosophila

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