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M RNA PROCESSINGPROKARYOTES AND EUKARYOTES

K.VIJAYREDDY

RNA (Ribonucleic Acid)

RNA is much more abundant than DNAThere are several important differences between RNA and DNA. The pentose sugar in RNA is ribose, in DNA it’s deoxyribose.In RNA, uracil replaces the base thymine (U pairs with A).RNA is single stranded while DNA is double stranded.

Primary structure of RNAA, C, G, and U are linked by 3’-5’ ester bonds between ribose and phosphate.

Secondary structure of RNA

D

A) Single stranded regionsformed by unpaired nucleotides B) Duplexdouble helical RNA (A-form with 11bp

per turn) C) Hairpinduplex bridged by a loop of unpaired nucleotidesD) Internal loopnucleotides not forming Watson-Crick base pairsE) Bulge loopunpaired nucleotides in one strand,other strand has contiguous base pairingF) Junctionthree or more duplexes separated by singlestranded regionsG) Pseudoknottertiary interaction between bases of hairpin loop and outside bases

G

AB

C

DE

F

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TERTIARY STRUCTUREPrimary: Covalent bonds & Secondary/Tertiary

Non-covalent bonds(H-bonds)

7

D D

obbs ISU - B

CB

444/544X: R

NA

Structure &

FunctionRNA STRUCTURE: 3 LEVELS OF ORGANIZATION

Rob KnightUniv Colorado

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RNA TYPES & FUNCTIONS Types of RNAs FunctionmRNA - messenger Template for protein synthesis.

rRNA - ribosomal Component of ribosome's (protein synthesis) .

t-RNA - transfer Transfer of amino acid (protein synthesis).

hnRNA - heterogeneous nuclear Precursors & intermediates of mature mRNAs & other RNAs (Premature mRNA).

scRNA - small cytoplasmic Signal Recognition Particle (SRP)tRNA processing .

snRNA - small nuclear snoRNA - small nucleolar

Participate in the splicing and transfer of hnRNA.rRNA processing/maturation/methylation.

miRNA-micro RNA Usually endogenous, induce degradation of targeted mRNA. that block expression of complementary mRNAs.regulation of transcription and translation.

siRNA-small interfering RNA Usually exogenous, induce degradation of targeted mRNA. regulation of transcription and translation.

ncRNA-non-coding RNA (npcRNA, nmRNA, fRNA)

all RNA other than mRNA,functional RNA molecule that is not translated into a protein. longer than 200nt.

Ribosome's are the sites of protein synthesis.They consist of ribosomal RNA (65%) and proteins (35%).They have two subunits, a large one and a small one.

Ribosomal RNA

Smallest RNAThe existence of tRNA was demonstrated by Hoagland in 1957.

Anticodon loop has 3 nucleotides that function as anticodon. .

Thymine loop function as the ribosome attachment region.

The DHU loop serves as the aminoacyl synthetase recognition region.

Base-pairing involving H bonds.

Transfer RNA(tRNA)

tRNA 3-D structuretRNA has a tertiary structure that is L-shaped

MESSENGER RNA (MRNA)

It is a single stranded base for base complementary copy of one DNA strand& provides information for amino acid sequence of a polypeptide.

Carries genetic information from nucleus to cytoplasm

(Template of protein synthesis).

Present in two forms: Active form: actively supports translation.

Inactive form: does not support translation..

Three main parts: 5’ untranslated region (5’ UTR) or leader

sequence

Coding sequence, specifies amino acids to be translated

3’ untranslated region ( 3’ UTR) or trailer sequence

These are catalytic RNAs that mainly participate in the cleavage of RNA.They are not true catalysts because they alter their own structure as a result of catalysis.Example: 1. RNase P is a common ribozyme that matures tRNA that acts as an endonuclease. 2. Self-splicing introns.

Clinical applications:-Used as therapeutic agents in correcting mutant mRNA in human cells and inhibiting unwanted gene expression.Kill cancer cells.Prevent virus replication.Gene inhibitors.Gene amenders.Protein inhibitors.Immuno stimulatory RNA’s.

Ribozymes

Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield

the mature product.(or)

RNA processing is the collective term used to describe the molecular events allowing the

primary transcripts to become the mature RNA.(or)

The process of modification, mainly through cleavage & or splicing, of primary RNA

transcripts so as to release functional RNA molecules from them.

It is carried out by ribonucleases (RNases) that cleave RNA.

What is RNA processing……!

These RNases not only process the RNA transcripts, also degrade the tRNA,

mRNA, rRNA, and other RNA molecules as a part of the normal cellular “Turnover

process”.

Turnover process refers to degradation of old molecules and the synthesis of new

molecules to replace them.

Both exo-and endo ribonuclease participate in the turnover process.

Primary transcript

Mature RNA.

Nucleus or NucleolusCytoplasm

RNA processing

Removal of nucleotides

addition of nucleotides to the 5’- or 3’- ends

modification of certain nucleotides

1)Remvoal of nucleotides by both endonucleases and exonucleases.

Endonucleases to cut at specific sites within a precursor RNA.

Exonucleases to trim the ends of a precursor RNA.

2)Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.

Add a cap and a poly(A) tail to pre-mRNA. AAAAAA3)Modification of certain nucleotides on either

the base or the sugar moiety. Add a methyl group to 2’-OH of ribose in

mRNA and rRNA.

Processing of mRNAhnRNPsnRNP particles5’Capping3’CleavagePolyadenylationSplicingPre-mRNA methylation

mRNA PROCESSING

Genetic information is transferred from genes to the proteins they encode via a “messenger” RNA

intermediate.

Characteristics of the Five RNA Polymerases of Eukaryotes

Enzyme Location Products

RNA polymerase I Nucleolus Ribosomal RNAs, excluding 5S rRNA

RNA polymerase II Nucleus Nuclear pre-mRNAs

RNA polymerase III Nucleus tRNAs, 5S rRNA, and other small nuclearRNAs

RNA polymerase IV Nucleus (plant) Small interfering RNAs (siRNAs)

RNA polymerase V Nucleus (plant) Some siRNAs plus noncoding (antisense)transcripts of siRNA target genes

MRNA PROCESSING IN PROKARYOTES

There is little or no processing of mRNA transcripts in prokaryotes. In fact, ribosomes can assemble proteins before mRNA molecules have not yet been completely synthesized.

Prokaryotic mRNA is degraded rapidly from the 5’-end therefore only be translated for a limited amount of time.

DNA

Cytoplasm

Nucleus

EUKARYOTIC MRNA TRANSCRIPTS ARE PROCESSED

ExportG AAAAAA

RNA

Transcription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA

The mRNA then moves out of the

nucleus and is translated in the

cytoplasm.

mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA).

Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs.

Pre-mRNA molecules are again processed to give mature mRNAs by 5’-capping, 3’-cleavage and poly adenylation, splicing and methylation.

Processing of mRNA

HNRNP:- HNRNA + PROTEINS

The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to

form heterogeneous nuclear Ribonucleoprotein (hnRNP).

The hnRNP proteins are keep the hnRNA in a single-stranded form and to assist in the various RNA

processing reactions.

SNRNP PARTICLES: SNRNA + PROTEINS

Eukaryotic nuclei contain many discreet small RNA species called small nuclear

RNAs(snRNAs) are rich in the base uracil, which complex with specific proteins to form

snRNPs.

The most abundant snRNP are involved in pre-mRNA splicing, U1,U2,U4,U5 and U6.

snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.

.

Removes the -phosphate

CAPPING – STEP 1

CAPPING –STEP 2

Attaches guanosine nucleotide

OHOH

OHOH

Adds methyl groups to guanine & riboses

CAPPING – STEP 3

OH OCH3

7-Methylguanine

5¢ 3¢

5¢

5¢ 3¢

Endonuclease cleavage occursabout 20 nucleotides downstreamfrom the AAUAAA sequence.

PolyA-polymerase addsadenine nucleotidesto the 3¢ end.

Polyadenylation sequence

PolyA tail

AAAAAAAAAAAA....AAUAAA

AAUAAA

AAUAAA

CLEAVAGE/POLYADENYLATION

G/U

SPLICING The process of cutting the pre-mRNA to

remove the introns and joining together of the exons is called splicing.

It takes place in the nucleus before the mature mRNA can be exported to the cytoplasm.

Most genes have their protein-coding information interrupted by non-coding sequences called “introns”. The coding sequences are then called “exons”.

Introns: non-coding sequences. Exons: coding sequences.

SPLICING BASED ON SECONDARY STRUCTURE:

SPLICEOSOME MEDIATED SPLICING: All the known introns begin with the

dinucleotide GT and end with the dinucleotide AG this is known as GT-AG rule

The GT dinucleotide depicts the donor splicing site and AG dinucleotide depicts the acceptor splicing site

SPLICING – INTRON SEQUENCES

3¢5¢5¢ splice site 3¢ splice siteBranch site

IntronExon ExonPy12N PyAGGA/CGGU Pu AGUA UACUUAUCC

Exon n ……A G G U A A G U …Intron …Y N Y Y R A Y …....Y12 N C A G N ….. Exon n+1 64 73 100 100 62 68 84 63 80 80 87 75 100 95 65 100 100

Branch Point

Yeast consensus

Intron loops out and exons brought closer together

SPLICING OF PRE-MRNAS

SPLICING OF PRE-MRNA

This is a Spliceosome

Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembled.

Intron will be degraded and the snRNPs used again

SPLICING OF PRE-MRNA

All noncoding introns are spliced out of a pre- mRNA by the Spliceosome.But not all exons are included in the final mRNA.mRNA can undergo alternative splicing.The selective inclusion or exclusion of exons occur.From one pre-mRNA can make many different mRNA(thus different proteins) >50% of human genes undergo alternative splicing.

ALTERNATIVE SPLICING

ALTERNATIVE SPLICING OF TROPOMYOSIN

* Alternative pA*

* Alternative pA*

HOWEVER, MULTIPLE INTRONS MAY BE SPLICED DIFFERENTLY IN DIFFERENT CIRCUMSTANCES, FOR EXAMPLE IN DIFFERENT TISSUES.

1 2 3 5Heart muscle mRNA

1 43 5Uterine muscle mRNA

Thus one gene can encode more than one protein. The proteins are similar but not identical and may have distinct properties.

This is important in complex organisms

3 5421pre-mRNA

Sex in Drosophila is largely determined

by alternative splicing

RNA EDITINGThis is a form of RNA processing in which

the nucleotide sequence of the primary transcript is altered by either

Changing residues,Deleting residues,Inserting residues at specific points

along the moleculeChanging RNA sequence (after

transcription).

Apolipoprotein-B mRNA in mammalian intestine and liver

The mammalian liver cells contain apolipoprotein- B having 4563 amino acids. While in intestine cells this protein has only 2152 amino acids.In intestine cells, the codon 2153 is modified in the mRNA , the C of this codon, CAA, is changed to U to give rise to the codon UAA, which causes chain termination. A guide RNA containing a sequence that is complementary to the correctly edited mRNA provides a mechanism of U insertion or deletion.

Liver Intestine

mRNA processing: overview

POSSIBLE QUESTIONS: Describe in detail about mRNA processing ? Difference between splicing and alternate

splicing? RNA editing?

REFERNCES: GENETICS- B.D.SINGH PRINCIPLES OF GENETICS- SNUSTARD AND SIMMONS

Thank

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