340 - 05 -- processing

Introns & Exons &

Alternative mRNA

Splicing; Oh My!!

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• 5’ untranslated region of mRNA can

switch between 2 conformations

• Antiterminator: allows transcription

to continue

• Terminator: stops transcription

• Switch is dependent on binding of a

small molecule to 5’ untranslated

region

Insect metamorphosis: regulated by ecdysone

Amphibian metamorphosis: regulated by thyroid hormone

Male vs. female development: regulated by testosterone and estrogens

All of these hormones have receptors that bind to DNA (zinc-finger proteins)

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Heritable changes not due to changes in DNA sequence itself (epi = besides or in addition to)

Chemical modification of bases• Cytosine methylation• After incorporation, DNA methylase adds methyl group

to 5 carbon• Methylation occurs more often if C is next to G• During replication, if both parent strands are

methylated, newly made strands will then be methylated by methylase

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Lots of methylation – lower transcription• X chromosome inactivation in female mammals

Accompanying video fair game

Transcriptional cosuppression• Extra copies of genes induce higher levels of

methylation (in all copies, not just the extra ones)

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Genetic imprinting• In germ line only (gamete forming cells)

• A few hundred genes are methylated

• Different methylation in females vs. males

• Imprinted genes are inactive during

embryogenesis (early development)

• Imprinting erased in germ cell development, re-

established later

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Insulator DNA sequences• Bind specific proteins

• Prevent enhancers from activating some

promoters

• Prevent chromatin condensation from interfering

with transcription

Acetylate nucleosomes on either side of actively

transcribed gene

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RNA selection: not all RNA is transported out of nucleus

RNA splicing• Translation doesn’t start at 5’ end of primary transcript

• Primary transcript includes: 5’ untranslated region

3’ untranslated region

Introns and exons (non-coding and coding regions)

Eukaryotes - must be processed prior to translation

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Three changes to primary transcripts

Two are “End modifications”• 1) 5’ “cap”

7- methylguanosine added to 5’ end

• 2) “Poly A tail”

Adenines added at 3’ end

The third is internal• 3) Splicing

Removes specific sequences from central part of

primary transcript

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5’ 3’

G-CapPoly-A tail

Coding regions are interrupted by noncoding segments - “split genes”

Coding regions - exons (ex for expressed)Noncoding regions - introns (intervening

sequences)Primary transcript contains exons and

introns RNA processing removes introns and

pastes exons together

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Number & size vary among genes &

species

Most eukaryotic genes have introns

Most of intron sequence may not be

important, but some sequence is important

Splice site sequences are important

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gene feature Median mean

Internal exon 122 bp 145 bp

# of exons 7 8.8

Size of introns 1023 bp 3356 bp

5’ untranslated 240 bp 300 bp

3’ untranslated 400 bp 770 bp

Coding sequence 1101 bp 1341 bp

Amino acids 367 bp 447 bp

Total length 14,000bp 27,000bp

14Don’t need to know this

Some genes have lots of introns and exons:

For example: Tropomyosin:• Structural protein associated with cytoskeleton

• Gene in mammals has 11 exons

Different tissues splice the exons together differently

Not all exons are used in some tissuesSo… resulting proteins also differ among

tissues

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Fig. 2.27Know what’s going on here

Different splicing results in different mRNA

Different mRNA’s produce different (although related) proteins

Proteins can vary in function

Common process (up to 75% of all genes may have alternative splicing)

Produce more proteins than you have genes

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mRNA stability: Longevity of mRNA determines how many proteins can be produced

• Deadenylation-dependent: enzymes trim poly-A tail, when poly-A tail is short enough (25-60 bases), mRNA can be “uncapped” which stops translation and leads to mRNA degradation

• Deadenylation-independent: decapping or endonuclease (enzyme cuts up mRNA) Common in mRNA’s with early stop codons, or unspliced

introns

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RNA interference (RNAi): post-

transcriptional silencing mechanism• Relies on production of double-stranded RNA’s

RNAi sequences complementary to short region of

mRNA

Double stranded RNA is recognized by cell and

enzymatically digested

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Germ line DNA has lots of unique sequences for variable regions of antibodies

Germ line DNA has lots of unique sequences for joining regions of antibodies

Splicing combines variable and joining regions randomly

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Results in lots of different combinations

Similar splicing occurs in DNA that specifies heavy chain of antibodies

Individual B cells differ in what combination of regions are spliced together

End result is that different antibodies are produced in different cells

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