RNA SPLICING - PowerPoint Presentation

Slide1

RNA SPLICING

SUBMITTED BY

SELMA ABDUL SAMAD

BCH-10-05-02

S3

MSc

BIOCHEMISTRYSlide2

Interrupted GenesProkaryotes – continuous gene(uninterrupted) Eukaryotes – gene is interrupted with non-coding sequences (introns)

RNA splicing

– the removal of these

introns

while joining the rest

Terminology

Exons

– sequences represented in mature RNA

(A gene starts and ends with

exons

that correspond to the 5’ and 3’ ends of RNA)

Introns

– Intervening sequences that are removed when the primary transcript is processed to the mature RNASlide3

DNA TRANSCRIPT(RNA COPY) MATURE RNASlide4

The mechanism excludes any splicing together of sequences representing different alleles A typical mammalian gene has 7 to 8 exons spread out over ~16 kb . The exons are relatively short(~100 – 200 bp) and introns

relatively long(>1 kb)

So, gene is interrupted while mRNA(~2.2 kb) is uninterrupted , which requires the primary transcript(

pre-mRNA

) to be processed.

Nuclear RNA(including pre-mRNA)

- much larger than mRNA

- very unstable

- much greater sequence complexity

- known as

hnRNA

(

heterogenous

nuclear RNA)Slide5

hnRNP – ribonuclear protein ; the physical form of hnRNA,which is bound to proteins ; Has the form of beads connected by a fiber.Splicing and other post transcriptional modifications take place in the nucleusSlide6
Slide7

SPLICING IS OF SEVERAL TYPESIn higher eukaryotes – introns removed by a system that recognizes only short consensus sequences conserved at exon-intron boundaries and within the

intron

.

- Requires

spliceosomes

(large splicing apparatus)

- Mechanism involves

transesterifications

- Catalytic center includes RNA and proteins.

Autonomous splicing

– of

introns

by certain RNAs

- 2 types of

introns

– distinguished by 2

0

and 3

0

structures

- Mechanism –

transesterification

- Catalytic agent – RNA (catalytic RNA)Slide8

Splicing of yeast tRNA – accomplished by enzymes that use cleavage and ligation.

SPLICE JUNCTIONS (

splic

e sites)

The

two

exon-intron

boundaries that include the sites of breakage and reunion.

ie

, the junction between

exons

and

introns

.

There is no extensive homology or

complementarity

between 2 ends of an

intron

. But there are well

conserved,short

,consensus

sequences.

High conservation is found only immediately within the

intron

at the presumed junctions.

Slide9

GT-AG rule – ie., GU-AG rule in pre-mRNA - An intron starts with dinucleotide GU and ends in AG

- called 5’ and 3’ splice sites resp.Slide10
Slide11

SPLICE JUNCTIONS ARE READ IN PAIRS In an mRNA, introns are multiple and longAppropriate 5’ and 3’ sites should be paired - It could be an intrinsic property of RNA to connect

the sites at the ends of a particular

intron

- Splicing could follow rules that ensure a 5’ site is

always paired to a following 3’ site

In principle any 5’ splice site can be connected to any 3’ splice site.

So,there

are preferred pathways that ensure right splicing.Slide12

The conformation of the RNA influences the accessibility of the splice sites. As particular introns are removed,the conformation changes and new pairs of splice sites become available.So , the splicing reaction does not proceed sequentially along the precursor RNA.Slide13

SPLICING PROCEEDS THROUGH A LARIATSplicing is independent of transcription or other post – transcriptional modifications,yet occur co-ordinated.In vivo, the

exons

are not released as free molecules during

splicing,but

remain held together by the splicing apparatus.

Splicing requires the 5’ and 3’ splice sites and a branch site just upstream of the 3’ splice site.

Steps in splicing

- A cut is made at the 5’ splice site, separating the left

exon

and the right

intron-exon

molecule

Slide14

SPLICING PROCEEDS THROUGH A LARIATSlide15

- The left exon becomes linear- The right intron-exon molecule form a lariat by forming a 5’-2’ bond between 5’ terminus and the target base ‘A’ called the branch site

- The 3’ splice site is then cut releasing free

intron

in the lariat form

- The right

exon

is

ligated

(

spliced

) to the left

exon

- The lariat is then

debranched

to give a linear excised

intron

which is rapidly degraded

The branch site plays an important role in identifying the 3’ splice site. The consensus is highly conserved in yeast as UACUAAC.Slide16

The branch site is not well conserved in higher eukaryotes, but has a preference for bases at each position and retains the target A nucleotide.The branch site lies 18 to 40 nucleotides upstream of the 3’ splice site.The lariat formation is effected by transesterification -

First, a

nucleophilic

attack by the 2’-OH of the invariant A on the 5’ splice site

- Second, the free 3’-OH of the

exon

that was released , now attacks the bond at the 3’ splice siteSlide17

THE SPLICING APPARATUSContains both proteins and RNAs ; Splicing occurs only after all components are sequentially assembled on the pre-mRNAThe small RNAs are found both in nucleus and cytoplasm of eukaryotic cellsIn nucleus – small nuclear RNAs (snRNAs)

In cytoplasm – small

cytoplasmic

RNAs (

scRNAs

)

In nucleolus –

snoRNAs

They exist as

ribonucleoprotein

particles

snRNPs

and

scRNPs

(known colloq. as

snurps

and

scyrps

)Slide18

Spliceosome – large particulate complex formed of snRNPs involved in splicing and many additional proteins - It comprises a 50S to 60S RNP particleSlide19

Like the ribosome, the spliceosome depends on RNA-RNA, protein-RNA and protein-protein interactions.The 5 snRNPs involved in splicing are U1, U2, U5, U4 and U6 . Each snRNP contains a single snRNA

and several(>20) proteins. U4 and U6 are usually found as a single U4/U6 particle.Slide20

SPLICEOSOME MACHINERYBefore any irreversible change is made to the RNA, all of the splicing components are assembled and have ensured that the splice sites are available.Splicing is divided into 2 stagesa) The 5’ splice site, branch sequence and adjacent pyrimidine tract are

recognised.The

spliceosome

complex is assembled

b) Structure of transcript is changed by cleavage and ligation. Components of the complex are released or

reorganised

as it proceeds through the reactions.Slide21

Binding of U1 snRNP to the 5’ splice site is the first step in splicing. ie.,one of its proteins,U1-70k interacts with protein ASF/SF2(an SR class general splicing factor) causing U1 snRNA to base pair with the 5’ site by a single stranded region at 5’ terminus (4 to 6 bases complementary with splice site).

Complementarity

between U1

snRNA

and 5’ splice site is necessary for splicing, with pairing stabilized by proteins of U1

snRNP

.

[

SR proteins – imp. group of splicing factors & regulators

- Take their name from Ser-

Arg

rich region with variable

length. They interact each other via these regions. They

bind RNA/connects U2AF to U1. They are essential part

of

spliceosome,forming

a framework on RNA substrate]Slide22
Slide23

The first complex formed during splicing is the E (early presplicing) complex – it contains U1 snRNP,U2AF(a splicing factor) and some SR proteins.The formation of E complex identifies a pre-mRNA as a substrate for formation of splicing complex and is hence also called the commitment complex.In the E complex, U2AF is bound to the region between the branch site and the 3' splice site. In most organisms, it has a large subunit (U2AF65) that contacts a

pyrimidine

tract downstream of the branch site; a small subunit (V2AF35) directly contacts the

dinucleotide

AG at the 3' splice site.Slide24

Another splicing factor, called SF1in mammals and BBP in yeast. connects V2AF/Mud2 to the U1 snRNP bound at the 5' splice site. Complex formation is enhanced by the cooperative reactions of the two proteins; SF 1 and U2AF (or BBP and Mud2) bind together to the RNA substrate -1 Ox more effectively than either alone. This interaction is probably responsible for making the first connection between the two splice sites across the intron.The E complex is converted to the A complex when U2 snRNP

binds to the branch site. Both UI

snRNP

and U2AF/Mud2 are needed for U2 binding. The U2

snRNA

includes sequences complementary to the branch site.Slide25

A sequence near the 5’ end of the snRNA base pairs with the branch sequence in the intron. Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site.The binding of U2 snRNP

requires ATP hydrolysis and commits a pre-mRNA to the splicing pathway by generating A

presplicing

complex.Slide26
Slide27

Formation of E complexIntron definition – The two splice sites are recognised without requiring any sequences outside of the

intron

.

The

SR proteins

may enable U2AFlU2

snRNP

to bind in

vitro in the

absence of UI, raising the possibility that there could be a U1-independent pathway for splicing

Exon

definition

– When

introns

are long and splice sites are weak ;

sequences downstream of the

intron

itself are required ; The 3‘ splice site is recognized as part of a complex that forms across the

next

exon

. though, in which the

next 5' splice site is also bound by UI

snRNA

. This UI

snRNA

is connected by SR proteins to the U2AF at the

pyrimidine

tract

.Slide28

5 snRNPs Form the SpliceosomeThe snRNPs and factors associate with E complex in a defined order.B1 complex – formed when a trimer

U5 and U4/U6 binds to A complex(U1 and U2

snRNPs

)

This is the

spliceosome

complex – has all components needed for splicing.

B2 complex – formed when U1

snRNA

is

released,other

components,esp

U6 comes into juxtaposition with 5’ splice site, and U5 shifts to the vicinity of

intron

sequences.Slide29
Slide30

The role of U4 snRNA may be to sequester U6 snRNA until it is needed. So U4 is released with hydrolysis of ATP ,triggering catalytic reaction.When U4 is released,the region of U6 initially base paired with U4 now is free. The first part of it pairs with U2; the second part forms an

intramolecular

hairpin.

Thus several pairing reactions between

snRNAs

and the substrate RNA occur in the course of splicing.Slide31

U6 snRNA is not used up in a splicing reaction and at completion must be released from U2 so that it can reform the duplex structure with U4 to undertake another cycle of splicing.In human genome,more than 98% introns are GU-AG .

Less than 1% are GC-AG

About 0.1 % are AU-AC type.

These

introns

required an alternate splicing apparatus that comprise the U12

spliceosome,containing

U11 , U12, a U5 variant and the U4

atac

and U6

atac

snRNAs

.

The splicing reaction is essentially similar to that of GU – AG

introns

.

ALTERNATE SPLICING APPARATUSSlide32

Some GU-AG introns may also be spliced by the U12 spliceosome and vice-versa.The two types of introns co-exist in a variety of genomes and may even be found in the same gene.

Introns

in protein coding genes are generally of 3 classes

* nuclear pre-mRNA

introns

* Group I

introns

* Group II

introns

Nuclear pre-mRNA

introns

are identified by the presence of GU-AG base sequence

AUTOSPLICINGSlide33

Group I and II introns are found in organelles and bacteria. Group I introns are more common.Each can be folded into a typical type of secondary structure.They have the ability to excise themselves from an RNA –

ie

autosplicing

. In

vivo,proteins

are required to assist folding.Slide34

All 3 classes of introns are excised by two successive transesterification reactions.There are parallels between group II introns

and pre-mRNA splicing. Group II mitochondrial

introns

are excised by the same mechanism as nuclear pre-mRNAs via a lariat that is held together by a 5'-2' bondSlide35
Slide36

The ability of group II introns to remove themselves by an autocatalytic splicing event stands in great contrast to the requirement of nuclear introns for a complex splicing apparatus. The snRNAs of the

spliceosome

as compensating for the lack of sequence information in the

intron

, and providing the information required to form particular structures in RNA and may have evolved from the autocatalytic system.Slide37

Thus the snRNAs may undergo reactions with the pre-mRNA substrate, and with one another, that have substituted for the series of conformational changes that occur in RNAs that splice by group II mechanisms.These changes have relieved the substrate pre-mRNA of the obligation to carry the sequences needed to sponsor the reaction. As the splicing apparatus has become more complex (and as the number of potential substrates has increased), proteins have played a more important role.Slide38

When an interrupted gene is transcribed into an RNA that gives rise to a single type of spliced mRNA, there is no ambiguity in assignment of exons and introns.But when a single gene gives rise to more than one mRNA sequence,it follows an alternative splicing pattern

.

In some cases, the ultimate pattern of expression is dictated by the primary transcript, because the use of different

startpoints

or the generation of alternative 3' ends alters the pattern of splicing.

ALTERNATIVE SPLICINGSlide39

In other cases, a single primary transcript is spliced in more than one way, and internal exons are substituted, added, or deleted.In some cases, the multiple products all are made in the same cell, but in others the process is regulated so that particular splicing patterns occur only under particular conditions.There is an ASF(Alternative Splicing Factor) which is same as that of the SF2 splicing factor.Both

are RNA binding proteins in the SR family.Slide40

sxl > traSlide41

When a pre-mRNA has more than one 5' splice site preceding a single 3' splice site, increased concentrations of ASF/SF2 promote use of the 5' site nearest to the 3' site at the expense of the other site. This effect of ASF/SF2 can be counteracted by another splicing factor, SF5.Alternative splicing may also be influenced by repression of one site.Slide42
Slide43
Slide44

TRANS-SPLICING REACTIONSIn genetic terms, splicing occurs only in cis. This means that only sequences on the same molecule ofRNA can be spliced together.Very rare and observed in vitro usually.

Seen in

vivo,in

some special situations.

When splicing occurs, a 5'-2' link forms by the usual reaction between the GU of the 5‘

intron

and the branch sequence near the AG of the 3'

intron

. The two parts of the

intron

are not covalently linked, and thus generate a

Yshaped

molecule instead of a lariat.Slide45
Slide46

The RNA that donates the 5' exon for transsplicing is called the SL RNA (spliced leader RNA) and exists as SLRNPsThe SL RNA can carry out the functions that the U1

snRNA

performs at the 5’ splice site.

The

trans-splicing reaction of the SL RNA

may represent a step toward the evolution of the pre-mRNA splicing apparatus.Slide47
Slide48

tRNA SPLICINGThe splicing of tRNA genes is achieved by a different mechanism that relies upon separate cleavage and ligation reactions.The introns

in

tRNA

genes representing different amino acids are unrelated.

There is no consensus sequence that could be recognized by the splicing enzymes.

All the

introns

include a sequence that is complementary to the

anticodon

of the

tRNA.This

creates an alternative conformation for the

anticodon

arm in which the

anticodon

is base paired to form an extension of the usual arm.Slide49

The exact sequence and size of the intron is not important.Splicing oftRNA depends principally on recognition ofa common secondary structure in

tRNA

rather than a common sequence

ofthe

intron

.

Regions in various parts of the molecule are important including the stretch between the acceptor arm and D arm, in the 1\If C arm, and especially the

anticodon

arm.Slide50

The two separate stages of the reaction are catalyzed by different enzymes. • The first step does not require ATP. It involves phosphodiester bond cleavage by an atypical nuclease reaction. It is catalyzed by an endonuclease.• The second step requires ATP and involves bond formation; it is a ligation reaction, and the responsible enzyme activity is described as an RNA

ligase

.

An

endonuclease

recognises

introns

and cleaves at both ends of the

introns

.Slide51
Slide52
Slide53
Slide54

SUMMARYSplicing accomplishes the removal of introns and the joining of exons into the mature sequence of RNA.The systems include eukaryotic nuclear

introns

, group I and group II

introns

, and

tRNA

introns

.

Each reaction is usually a

cis

-acting event.

Consensus sequences

GU-AG rule

Transesterification

and lariat formation

Spliceosome

formation,

Autosplicing

, Alternative Splicing, Trans splicing ,

tRNA

splicingSlide55

REFERENCEBenjamin Lewin,GENES IX , pg.667 – 695Watson,Baker et.al , Molecular Biology of the Gene,pg

. 379 - 409Slide56

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