Download RNA Splicing

Interrupted Genes
• Prokaryotes – 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 RNA
DNA
TRANSCRIPT(RNA COPY)
MATURE RNA
• 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)
• 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 nucleus
SPLICING IS OF SEVERAL TYPES
• In 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 20 and 30
structures
- Mechanism – transesterification
- Catalytic agent – RNA (catalytic RNA)
• Splicing of yeast tRNA – accomplished by enzymes that
use cleavage and ligation.
SPLICE JUNCTIONS (splice 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.
• 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.
SPLICE JUNCTIONS ARE READ IN PAIRS
• In an mRNA, introns are multiple and long
• Appropriate 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.
• 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.
SPLICING PROCEEDS THROUGH A LARIAT
• Splicing 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
SPLICING
PROCEEDS
THROUGH A
LARIAT
- 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.
• 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 site
THE SPLICING APPARATUS
• Contains both proteins and RNAs ; Splicing occurs only
after all components are sequentially assembled on the
pre-mRNA
• The small RNAs are found both in nucleus and
cytoplasm of eukaryotic cells
In 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)
• Spliceosome – large particulate complex formed of
snRNPs involved in splicing and many additional proteins
- It comprises a 50S to 60S RNP particle
• Like the ribosome, the spliceosome depends on RNARNA, 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.
SPLICEOSOME MACHINERY
• Before 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 stages
a) 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.
• 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]
• 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.
• 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.
• 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.
Formation of E complex
• Intron 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.
5 snRNPs Form the Spliceosome
• The 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.
• 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.
• 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.
ALTERNATE SPLICING APPARATUS
• 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 U4atac and U6atac snRNAs.
• The splicing reaction is essentially similar to that of GU –
AG introns.
• 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.
AUTOSPLICING
• 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
• 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.
• 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' bond
• 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.
• Thus the snRNAs may undergo reactions with the premRNA 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.
ALTERNATIVE SPLICING
• 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.
• 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.
•
sxl > tra
• 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.
TRANS-SPLICING REACTIONS
• In 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.
• The RNA that donates the 5' exon for transsplicing is called
the SL RNA (spliced leader RNA) and exists as SLRNPs
• The 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.
tRNA SPLICING
• The 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.
• 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.
• 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.
SUMMARY
• Splicing 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 splicing
REFERENCE
• Benjamin Lewin,GENES IX , pg.667 – 695
• Watson,Baker et.al , Molecular Biology of the
Gene,pg. 379 - 409