mRNA

1
mRNA

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Review
• mRNA molecules
• Modification and processing
• Chemical modification of 5 and 3 ends
• Intron splicing
• RNA editing (insertion, changing, deleting)

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Review

• 5 CCUAGUAUUCAGUCCGCCAUGCG GACAAGCUACUCCA 3

5 UACU 3 3 AUGA 5
5 GUCCG 3 3 CAGGC 5
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mRNA

• Whats the deal with mRNA?

Early work

• Genetic information is on DNA in nucleus

• Genes code for proteins

• Protein synthesis occurs in cytoplasm

Therefore, there must be a mechanism for carrying
information within the cell.
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mRNA

• The messenger molecule is RNA (mRNA)

• Polymer of ribonucleotides (like other RNAs)

• Physically caries coded genetic information from
  DNA to the ribosomes as instructions for protein
  synthesis

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mRNA

• Turnover rate

• Unlike tRNA and rRNA, mRNA is unstable

• mRNA half-life is only minutes or hours
• Half-life time when half of mRNAs are destroyed

• mRNA half-life is consistent level of mRNA in
  the cell is related to transcription rate.

Therefore, rate of transcription controls how
much protein is produced.
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Modification of mRNA

• Prokaryotic vs. Eukaryotic

• Prokaryote mRNA goes to ribosome as direct copy
  of the gene

• Eukaryote mRNA is modified before it goes to
  ribosome

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Modification of mRNA (eukaryotes)

• Types of modification

• Modification of two ends of mRNA

• Removal of Introns

• Altering sequence of mRNA

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Modification of mRNA (eukaryotes)

• Modifying ends of mRNA

• 5 ends are capped (m7G)

• 3 ends are polyadenylated (AAAAAAAA)

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Modification of mRNA (eukaryotes)

• Capping mRNA

• All eukaryotic mRNAs are capped

• Unmodified mRNA has triphosphate 5 end
• pppAGCAUCGAUG

• Mature mRNA has 7-methylguanine cap
• m7GpppAGCAUCGAUG

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Modification of mRNA (eukaryotes)

• Capping mRNA

5 A U C G A U C G A U C
5 pppApUpCpGpApUpCpGpApUpC
5m7GpppApUpCpGpApUpCpGpApUpC
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Modification of mRNA (eukaryotes)

• 7-methylguanine

Sugar
Guanine
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Modification of mRNA (eukaryotes)

• 7-methylguanine

CH3

Sugar
7-methylguanine
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Modification of mRNA (eukaryotes)

• Capping mRNA - How

• Guanine added by an unusual 5-5 bond

• Triphosphate link as opposed to 1 phosphate

• Methylation occurs after addition of guanine

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Modification of mRNA (eukaryotes)

• Capping mRNA Why?

• No definite reason for capping is known

• Probably plays role in translation

Duh!
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Modification of mRNA (eukaryotes)

• Polyadenylating mRNA

• Most eukaryotic mRNA is polyadenylated at 3 end
  of mature molecule

• Does not occur at 3 end of primary transcript

• Primary transcript is first cut between signal
  sequence and GU-rich sequence

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Modification of mRNA (eukaryotes)

• Polyadenylating mRNA

• Primary transcript is first cut between signal
  sequence and GU-rich sequence

• Signal sequence 5-AAUAAA-3
• GU-rich sequence rich in Gs and Us
• Signal sequence is upstream from GU-rich
• 30-40 base pairs between these regions

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Modification of mRNA (eukaryotes)

• Polyadenylating mRNA How?

• Proteins bind at signal sequence and GU-rich
  sequence

• mRNA is cut between

• Poly(A)polymerase adds As to end

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Modification of mRNA (eukaryotes)
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Modification of mRNA (eukaryotes)

• Polyadenylating mRNA - Why

• No reason for polyadenylation is known

• Remember some do not have polyA tail

• Perhaps the length of the poly A tail relates to
  how long the mRNA survives

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Modification of mRNA (eukaryotes)

• Polyadenylating mRNA

See Purification of mRNA (handout)
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Modification of mRNA (eukaryotes)

• Intron Splicing

Intergenic DNA
3
5
5
Gene 1
Gene 2
3
3
5
5
Gene 1
Gene 2
3
Read, but cut out later
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Modification of mRNA (eukaryotes)

• Intron Splicing

INTRON
5
3
EXON
EXON
Gene
INTRON
5
3
EXON
EXON
After Transcription Before Splicing
5
3
EXON
EXON
After Splicing Before Translation
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Modification of mRNA (eukaryotes)

• Intron Splicing

AGCTACTTGTATCGATAGATAGCATCGATTA
5
3
Gene
AGCUACUUGUAUCGAUAGAUAGCAUCGAUUA
5
3
After Transcription Before Splicing
AGCUACUUCAUCGAUUA
5
3
After Splicing Before Translation
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Modification of mRNA (eukaryotes)

• Intron Splicing (5 types of intron)

• GT-AG introns
• Self splicing intron
• AT-AC introns
• Group II introns
• tRNA introns

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Modification of mRNA (eukaryotes)

• Intron Splicing (5 types of introns)

• GT-AG introns
• Self splicing intron
• AT-AC introns
• Group II introns
• tRNA introns

Almost all mRNA introns are GT-AG introns.
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Modification of mRNA (eukaryotes)

• GT-AG Introns

• Have GT after first splice site and AG before
  second splice site (within gene)

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Modification of mRNA (eukaryotes)

• GT-AG Introns

mRNA
AGAGGUAAGUAUCUCCCUACAGCAUCGAUUA
5
3
After Transcription Before Splicing
AGAGGUAACAUCGAUUA
5
3
After Splicing Before Translation
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Modification of mRNA (eukaryotes)

• GT-AG Introns

Splice Sites
mRNA
AGAGGUAAGUAUCUCCCUACAGCAUCGAUUA
5
3
After Transcription Before Splicing
AGAGGUAACAUCGAUUA
5
3
After Splicing Before Translation
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Modification of mRNA (eukaryotes)

• GT-AG Introns

• Have GT after first splice site and AG before
  second splice site (within gene)

• Actual sequences are more complex

• 5 splice site 5 AGGTAA GT 3

• 3 splice site 5 YYYYYYNCAG 3

• Sequences may vary slightly

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Modification of mRNA (eukaryotes)

• GT-AG Introns

mRNA
AGAGGUAAGUAUCUCCCUACAGCAUCGAUUA
5
3
After Transcription Before Splicing
AGAGGUAACAUCGAUUA
5
3
After Splicing Before Translation
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Modification of mRNA (eukaryotes)

• GT-AG Introns

• snRNAs are involved with splicing
• Small nuclear RNA (not snoRNA)
• Uracil rich approximately 100-200 nucleotides
• examples U1,U2,U3,U4,U5,U6

• snRNP snRNAs proteins complex
• small nuclear ribonucleoproteins
• same name as the snRNA it contains

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Modification of mRNA (eukaryotes)

• GT-AG Introns How?

• Cleavage at the 5 splice site
• Free 5 end of intron attached to internal site
  of intron to form a lariat. (always attaches to
  an A)
• 3 splice site is cleaved
• Two exons join together

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Modification of mRNA (eukaryotes)

• GT-AG Introns How?

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Modification of mRNA (eukaryotes)

• GT-AG Introns How?

• Actual mechanism involves clumping of snRNAs to
  form a complex called the spliceosome

• Spliceosome cuts and joins mRNA, but none of the
  proteins in the spliceosome have been shown to
  individually have the necessary activity.

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Modification of mRNA (eukaryotes)
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Modification of mRNA (eukaryotes)

• Self-splicing Intron (Group I)

• Found in rRNA genes of certain protozoa

• Does not have consensus sequence or spliceosome

• Intron folds up, cleaves, and rejoins itself in
  the complete absence of protein

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Modification of mRNA (eukaryotes)

• Other Introns

• AT-AC similar to GT-AG

• Group II lariat with some self splicing activity

• tRNA introns short, in anticodon loop. Their
  removal is similar to process of cutting tRNA
  from tRNA precursor

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Modification of mRNA (eukaryotes)

• RNA Editing

• Alteration of the RNA sequence following
  transcription.

Studied example human apolipoprotien-B

• In liver, mRNA is translated normally
• In intestines, a specific C is converted to U
• CAA (glutamine) to UAA (terminator)

Results in shorter protein in the intestine
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Modification of mRNA (eukaryotes)

• RNA Editing

• Conversion is by a cytidine deaminase enzyme that
  recognizes target sequences on both sides of the
  C to be changed