post transcriptional modifications

POST TRANSCRIPTIONAL MODIFICATIONS

Prepared by: Narasimha Reddy.P.K

(2014-11-104)college of horticulture

kerala agricultural universityVellanikkara,thrissur

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Eukaryotic vs. Prokaryotic Transcription

• In eukaryotes, transcription and translation occur in separate

compartments.

• In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is

usually monocistronic.

– Polycistronic: one mRNA codes for more than one polypeptide

– monocistronic: one mRNA codes for only one polypeptide

• “Processing” of mRNA is required in eukaryotes for the

maturation

• No processing in prokaryotes(mRNA matures on transcription)

Coupled transcription and

translationmRNA processed and transported

out of nucleus for translation

Introduction…

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DNA

Transcription

Pri -RNA

tRNA

mRNA

rRNAP

roce

ss

ing

Introduction

Pri - transcript

Matured RNAs

• Capping (addition of

a 5’ 7-methyl guanosine

cap)

•Splicing to remove

intervening sequences

(introns)

• Polyadenylation

(addition of a poly-A

tail at the 3’)

1. mRNA Processing

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5’Capping

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Guanyltransferase

O-methyl transferase

Pri-mRNA

Cap Functions

Cap provides:

1. Protection from some ribonucleases degradation

2. Stabilizes mRNA

3. Enhanced translation and splicing

4. Enhanced transport from nucleus to cytoplasm

• mRNA is called hnRNA (heterogenous nuclear RNA) before splicing

occurs

•The hnRNP proteins to help keep the hnRNA in a single-stranded form

and to assist in the various RNA processing reactions

• Exon and intron lengths & numbers vary in various genes

• Exon (Expressed sequences)is any segment of an interrupted gene

that is represented in the mature RNA product.

• Intron (intervening sequences )is a segment of DNA that is

transcribed, but removed from within the transcript by splicing

together the sequences (exons) on either side of it.

mRNA splicing

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Splice Junction Consensus Sequence

• GU-AG rule describes the presence of these constant dinucleotides at the first

two and last two positions of introns of nuclear genes.

• Splice sites are the sequences immediately surrounding the exon-intron boundaries

• Splicing junctions are recognized only in the correct pairwise combinations

The sequence of steps in the production of mature eukaryotic mRNA as shown for the chicken ovalbumin gene.

• Splicing is mediated by a large RNPs(Ribonucleoproteins)

complex spliceosome

• Spliceosome contains a specific set of base Uracil-rich

snRNPs (small nuclear RNPs) associated with proteins

(snRNA complex with protein)

Function of snRNPs:

• Recognizing the 5’ splice site and the branch site.

• Bringing those sites together.

• Catalyzing (or helping to catalyze) the RNA cleavage.

mRNA splicing

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Spliceosome Complex• Splicing snRNPs:

• U1: 5'- site recognition

• U2: branch site recognition

• U4: forms base pairedcomplex & acts with U6

• U5: 3'- junction binding ofU4-U6 complex

• U6: complex with U4 makesspliceosome transesterase

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spliceosomes recognize introns starting with 5'-GU and ending in AG-3’

U1

3′5′

5′ splice site 3′ splice siteBranch site

AGU

Exon 1 Exon 2

U1 binds to 5′ splice site.U2 binds to branch site.

AG

3′5′

A

U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together.

U1 snRNP

U2 snRNP

3′5′

A

U5 snRNP

U4/U6 snRNP

U2

Intron loops out and exons brought

closer together

Mechanism of Spliceosome

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U1U4

3′5′

3′5′

5′ splice site is cut.

5′ end of intron is connected to the

A in the branch site to form a lariat.U1 and U4 are released.

3′ splice site is cut.

Exon 1 is connected to exon 2.

The intron (in the form of a lariat) is released along with U2, U5, and U6 (intron will be degraded).

A

A

U5U6

U5U6

U2

Intron plus U2,U5, and U6

Two connectedexonsExon 1 Exon 2

U2

Intron will be degraded and the snRNPs used

again

Mechanism of Spliceosome

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pre-mRNA are spliced in several different ways, allowing a single

gene to code for multiple proteins

The generation of different mature mRNAs from a particular type

of gene transcript can occur by varying the use of 5’- and 3’- splice

sites

Alternative splicing

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Sex determination in the Drosophila

Polyadenylation

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Polyadenylation Complex

Consensus sequence for 3’

process

Polyadenylation of mRNA at the 3’ end

CPSF: cleavage and polyadenylation specificity factor

binds upstream AAUAAA poly(A) Signal 5’ end.

CStF: cleavage stimulatory factor F interacts with a

downstream GU- sequence & bound with CPSF

forming a loop in RNA

CFI & CFII: cleavage factor I & II.

PAP: poly(A) polymerase stimulates cleavage at poly A

site

Bound PAP adds ≈12 A residues at a slow rate to 3’-

OH group

PABPII: poly(A)-binding protein II.

PABPII (short poly A tail) accelerates rate of addition

of A by PAP

After 200–250 A residues have been added, PABPII

signals PAP to stop polymerization

Poly (A) tail controls mRNA stability & influences

translation

• 5’ cap

• 5’ untranslated region

• Start codon

• Coding sequence

• Stop codon

• 3‘ untranslated region

• Poly A tail

Matured mRNA

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• Smallest among RNAs (75-93 nucleotides)

• Recognizes codon on mRNA

• Shows high affinity to amino acids

• Carry amino acids to the site of protein synthesis

• tRNA is transcribed by RNA polymerase III

• tRNA genes also occur in repeated copies

throughout the genome, and may contain introns.

2. tRNA

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Transfer RNA/ Soluble RNA/ supernatant RNA/ Adaptor

RNA

1. Removal of leader sequence &

trailer

2.Replacement of nucleotide

3.Modification of certain bases:

• Replacement of U residues at the

3′ end of pre-tRNA with a CCA

sequence

• Addition of methyl and

isopentenyl groups to the

heterocyclic ring of purine bases

• Methylation of the 2′-OH group

in the ribose of any residue; and

conversion of specific uridines to

dihydrouridine(D),pseudouridine(y)

4.Excision of an intron

Processing of tRNA

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Ribozyme

RNA can act as an Enzyme and catalyse reactions including its own replication

tRNA PROCESSING AND MATURATION

• In cell >80% of rRNA

• Serves to release mRNA from DNA

•Act as ribozymes in protein synthesis

• Relatively G:::C rich

• Ribosome

• Prokaryotes – 70S (50S & 30S)

• Eukaryotes – 80S (60S & 40S)

• Prokaryotes – In 50S subunits - 23S & 5S :31 proteins

In 30S subunits - 16S :21 proteins

• Eukaryotes – In 60S sub-units – 28S, 5.8S and 5S :50 proteins

In 40S sub-units – 18S :33 proteins

3. Ribosomal RNA (rRNA)

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Processing of ribosomal RNA

• Processing of 45s molecules occurs inside nucleolus

• 45s molecules tightly associated protein forming (RNPs)

• Frist cleavage: occurs at site I & remove 5’ terminal leader

sequence, produces 41s intermediate & 18s

• Second cleavage: occurs 41s intermediate at site 3’

generates 32s intermediate

• Final cleavage: separation of 32s intermediate into 28s, 5.8s

• Processed rRNA 28s, 5.8s & 18s

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2nd Cleavage

Processing of ribosomal RNA

Processing of ribosomal RNA

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Synthesis of 5S rRNA

• rDNA cistron for 5S rRNA is present outside Nucleolar

organizer

• Transcription requires RNA pol III + TFIIIA, TFIIIB &

TFIIIC