rna processing and rnps section o molecular biology course

RNA PROCESSING AND RNPs

Section O

•Molecular Biology Course

RNA Processing Very few RNA molecules are

transcribed directly into the final mature RNA.

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

RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

primary transcriptprimary transcript

mature RNAmature RNA..

Nucleus or Nucleolus

Cytoplasm

RN

A

pro

cessin

g

Romoval of nucleotides

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

modification of certain nucleotides

(1) Removal of nucleotides by both endonucleases and exonucleases

endonucleasesendonucleases to cut at specific to cut at specific sites sites withinwithin a precursor RNAa precursor RNA

exonucleasesexonucleases to trim the to trim the endsends of a of a precursor RNAprecursor RNA

This general process is seen in This general process is seen in prokaryotes and eukaryotes for all prokaryotes and eukaryotes for all types of RNAtypes of RNA

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

Add a cap and a Add a cap and a poly(A)poly(A) tail to pre- tail to pre-mRNAmRNA

(3) Modification of certain nucleotides on either the base or the sugar moiety.

–Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA

–Extensive changes of bases in tRNA

RNPsRibonucleoproteins =

RNA protein complexes

The RNA molecules in cells usually The RNA molecules in cells usually exist complexed with proteinsexist complexed with proteins

specific proteins attach to specific specific proteins attach to specific RNAsRNAs

RibosomesRibosomes are the largest and most are the largest and most complex RNPscomplex RNPs

3-D structure

Digital cryo-electron micrographyRN

P

颗粒的低温电镜

图

O1: rRNA PROCESSING AND RIBOSOMESO1: rRNA PROCESSING AND RIBOSOMES rRNA processing in prokaryotes rRNA processing in eukaryotesProkaryotic ribosomesEukaryotic ribosomes

Section O: RNA processing and RNPs

O1-1: rRNA processing in prokaryotes1. There are 7 different operons for rR

NA that are dispersed throughout the genome.

2. Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.

3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived(fig1)

rRNA operonrRNA operon

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA

Pre-tRNA

Promoters Terminators

O1-1: rRNA processing in prokaryotes


RNase III, involved in the first step of rRNA processing

RNase M5, M16 and M23 are involved in the second step of rRNA processing

30S pre-rRNA

Transcription

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA

Pre-tRNA


Processing steps


Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences

RNA folding

Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome

RNP complex formation

Step 3: After the binding of proteins, nucleotide modifications take place.

Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM)

Step 4: RNA cleavage

O1-1: rRNA processing in prokaryotes: CLEAVAGE

Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA Pre-tRNA


30S pre-rRNA: Transcription

Cleavage at

16S rRNA tRNA 23S rRNA 5S rRNA tRNA

RNase III III P F III III P F P ERNase III III P F III III P F P E

RNase M16 M16 M23 M23 M5RNase M16 M16 M23 M23 M5

rRNA operonrRNA operon

O1-2: rRNA processing in eukaryotesrRNA in eukaryotes is also

generated from a single, long precursor molecule by specific modification and cleavage steps

The processes are not so well understood

1. The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I

2. Precursor sizes are different among organisms (yeast: 7000 nt; mammalian 13500 nt), and pre-mRNA processing is also slightly different among organism.

O1-2: rRNA processing in Eukaryotes: rRNA features

3. The precursor contains one copy of the 18S coding region and one copy each of the 5.8S and 28S coding

regions, which together are the equivalent of the 23S rRNA in prokaryote

4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.


5. The eukaryotic 5S rRNA is transcribed by RNA Pol III from

unlinked genes to give a 121nt transcript

the transcript undergoes little or no processing


18S rRNA 5.8S rRNA 28S rRNA

45S

41S

20S and 32S

Mature rRNAs

47S18S 5.8S 28S

ETS1 ITS1 ITS2 ETS2

Indicates RNase cleavage

Mammalian pre-rRNA processingMammalian pre-rRNA processing

The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced.

Mature rRNAs complex with protein to form RNPs (nucleolus)

Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

O1-2: rRNA processing in Eukaryotes: rRNA processing

Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs.

Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

O1-2: rRNA processing in Eukaryotes: rRNA processing

Protein biosynthetic machinery

Made of 2 subunits (bacterial 30S and 50S, & Eukaryotes 40S and 60S)

O1-3: Ribosomes

Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes

In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.

Prokaryotic Ribosome

E. coli ribosome is 25 nm in diameter, 2750 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+

Prokaryotic Ribosomal proteins1. Size varied from 46 aa to 557 aa.

2. Most are basic proteins which bind to RNA.

3. rRNA chaperons to assist the folding of rRNA to catalytic structure.

Ribosomal rRNAResponsible for peptide bond format

ion.


Features of the E.coli ribosome

Cleft

PlatformCentral protuberance

Small

Stalk

Ribosome Assembly

Assembly is coupled w/ transcription and pre-rRNA processing


Ribosome Structure (1)


mRNA is associated with the 30S subunit

Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits.

The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.

Ribosome Structure (2)

Eukaryotic Ribosome larger and more complex than prokaryotic

ribosomes, but with similar structural and functional properties

O2: tRNA PROCESSING, RNase P AND RIBOZYMES tRNA processing in prokaryotes tRNA processing in eukaryotesRNase PRibozymes


tRNA 3-D structure

tRNA processing in prokaryotes Mature tRNAs are generated by

processing longer pre-tRNA transcripts, which involves

1. specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by

2. base modifications which are unique to each particular tRNA type.

tRNA processing in prokaryotes

Primary transcripts

RNase D,E,F and P(See your text book)

tRNA with mature ends

Base modifications

mature tRNAs

tRNA processing in eukaryotes

The pre-tRNA is synthesized with a

1. 16 nt 5’-leader,

2. a 14 nt intron and

3. two extra 3’-nucleotides.

1. Primary transcripts forms secondary structures recognized by endonucleases

2. 5’ leader and 3’ extra nucleotide removal

3. tRNA nucleptidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end

4. Intron removal

tRNA processing in Eukaryotes

RNase P

Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors

RNase P (2) RNase P enzymes are found in both

prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)

In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.

RNase P (1)

RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

Ribozyme (1)

Ribozymes are catalytic RNA molecules that can catalyze particular biochemical reactions.

RNase P RNA is a ribozyme. RNase P RNA from bacteria is more c

atalytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.

Ribozyme (2)

Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences

1. Group I introns, such as Tetrahymena intron

2. Group II introns.

Ribozyme (3)

Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA

1. HDV ribozyme2. Hairpin ribozyme3. Hammer head ribozyme

Ribozyme (4)

Ribozymes can be used as therapeutic agents in

1. correcting mutant mRNA in human cells

2. inhibiting unwanted gene expression

Kill cancer cells Prevent virus replication

O3: mRNA PROCESSING, hnRNPs AND snRNPs Processing of mRNA hnRNP snRNP particles 5’Capping 3’Cleavage and polyadenylation Splicing Pre-mRNA methylation


Processing of mRNA: prokaryotes

There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.

Prokaryotic mRNA is degraded rapidly from the 5’ end

Processing of mRNA in eukaryotes

In eukaryotes, 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 processed to mature mRNAs by 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation.


Eukaryotic mRNA processing: overview

hnRNP: hnRNA + proteins The hnRNA synthesized by RNA Pol I

I is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)

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

snRNP particles: snRNA + proteins

1. snRNAs are rich in the base uracil, which complex with specific proteins to form snRNPs.

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

3. A large number of snRNP define methylation sites in pre-rRNA.

snRNP particles

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

Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.

Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.

5’ Capping Very soon after RNA Pol II starts ma

king a transcript, and before the RNA chain is more then 20 -30 nt long, the 5’-end is chemically modified.

7-methylguanosine is covalently to the 5´ end of pre-mRNA.

Linked 5´ 5´ Occurs shortly after initiation


7-methylguanosine (m7G)

Function of 5´cap

Protection from degradation Increased translational efficiency Transport to cytoplasm Splicing of first exon

3’ Cleavage and polyadenylation

In most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.


RNA polymerase II does not usually terminate at distinct site

Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)

~250 AMPs are then added to the 3´ end

Almost all mRNAs have poly(A) tail

Function of poly(A) tail

Increased mRNA stability Increased translational efficiency Splicing of last intron

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.


Introns: non-coding sequences Exons: coding sequences RNA splicing: removal of introns

and joining of exons Splicing mechanism must be

precise to maintain open reading frame

Catalyzed by spliceosome (RNA + protein)


Biochemical steps of pre-mRNA splicing


Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site

Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.

C U R A Y

Lariat

Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

Spliceosome Catalyzes pre-mRNA splicing in nucleus Composed of five snRNPs (U1, U2, U4, U5

and U6), other splicing factors, and the pre-mRNA being assembled

U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.

U2 and U6 snRNA are able to catalyze the splicing reaction.


Splicing cycle

Pre-mRNA methylation

The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.

The methylations seem to be largely conserved in the mature mRNA.


O4: ALTERNATIVE mRNA PROCESSING

Alternative processing

Alternative poly(A) sites

Alternative splicing

RNA editing

Alternative processing

Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.

Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

Alternative poly(A) sites

Some pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.

In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

Alternative splicing The generation of different mature m

RNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways:

(i) By using different promoters(ii) By using different poly(A) sites(iii) By retaining certain introns(iv) By retaining or removing certain exo

ns

Alternative splicing

(A) A cassette exon can be either included in the mRNA or excluded.

(B) Mutually exclusive exons occur when two or more adjacent cassette exons are spliced such that only one exon in the group is included at a time.

(C, D) Alternative 5’ and 3’ splice sites allow the lengthening or shortening of a particular exon.

(E, F) Alternative promoters and alternative poly(A) sites switch the 59- or 39-most exons of a transcript.

(G) A retained intron can be excised from the pre-mRNA or can be retained in the translated mRNA.

(H) A single pre-mRNA can exhibit multiple sites of alternative splicing using different patterns of inclusion.

Sex in Drosophila is largely

determined by alternative

splicing

Sxl-Sex lethal gene: the master regulatory gene at the top of the sex determination pathway. The downstream targets of the Sxl protein include transcripts from the Transformer (Tra) and Male-specific lethal 2 (Msl2) genes. Tra gene also encodes a splicing regulator.

RNA editing

An unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing.

Changing RNA sequence (after transcription)

RNA editing is known to occur in two different situations, with different causes. In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. (Base modification:A or C deamination) In trypanosome mitochondria, more widespread changes occur in transcripts of several genes, when bases are systematically added or deleted. (Base U insertion and deletion)

1. Apolipoprotein-B mRNA in mammalian intestine and liver. The genome contains a single (interrupted) gene whose sequence is identical in all tissues, with a coding region of 4563 codons.

• a protein of 512 kD representing the full coding sequence is found in the liver.

• A shorter form of the protein, ~250 kD, is synthesized in intestine. This protein consists of the N-terminal half of the full-length protein, which is caused by the change of a CAA codon to a UAA. (see Figure)

Deamination in mammalian

Liver Intestine

Deamination in mammalian

2. glutamate receptors in rat brain. • Editing at one position changes a glutamine codon in DNA into a codon for arginine in RNA (AI)• The change affects the conductivity of the channel and therefore has an important effect on controlling ion flow through the neurotransmitter.

Deamination is catalyzed by cytidine or adenosine deaminases.Editing enzymes are related to the general deaminases, but have other regions or additional subunits that control their specificity.

Insertion or deletion of U in protozoa

Example: part of the mRNA sequence of Trypanosome brucei coxIII shows many uridines that are not coded in the DNA (shown in red) or that are removed from the RNA (shown as T).

Insertion or deletion of U in protozoa

A guide RNA containing a sequence that is complementary to the correctly edited mRNA provides a mechanism of U insertion or deletion. (See figure)

rna processing and rnps section o molecular biology course

Documents