Transcript MBch13(2008)

Chapter 13 RNA splicing
•
•
•
•
•
•
•
The chemistry of RNA splicing
The spliceosome machinery
Splicing pathways
Alternative splicing
Exon shuffling
RNA editing
mRNA transport
The chemistry of RNA splicing
Sequences within the RNA determine
where splicing occurs
The intron is removed in a form called a lariat as the
flanking exons are joined
Fig 13-5 The structure of
the three-way junction
formed during the
splicing reaction
•
The splicing reactions have no net gain in the no. of
chemical bonds. Yet, a large number of ATP is consumed,
not for the chemistry, but to properly assemble and
operate the slicing machinery.
•
What ensures the slicing only goes forward?
(1) Increase in entropy
(2) Excised intron is quickly degraded
Box 13-1 Adenovirus and the discovery of splicing
Map of the human adenovirus-2 genome
Human adenovirus is a DNA virus which serve as a model for studying
eukaryotic gene regulation.
Same promoter and the tripartate leader sequence for all the transcripts which
are resulted from alternative splicing
•R-loop mapping of the adenovirus-2 late messenger RNAs.
Exons from different RNA molecules can be fused by trans-splicing
Fig 13-6 trans-splicing
Although generally rare,
trans-spicing occurs in
almost all the mRNAs of
trypanosomes (錐體蟲)
eg. Nematode worms
(C. elegans)
The spliceosome machinery
• RNA splicing is carried out by a large complex called the
spliceosome
• Spliceosome comprises about 150 proteins and 5 RNAs
and is similar in size with ribosomes.
• Many functions of spliceosome are carried out by RNAs
rather by proteins.
• Five RNAs (U1, U2, U4, U5 and U6) are collectively
called small nuclear RNAs (snRNAs) with the size in the
range of 100-300 bp long and are complexed to
• small nuclear ribonuclear proteins (snRNP)
• The makeup of spliceosome varies at different stages of the
splicing reaction.
Fig 13-7 some RNA-RNA hybrids formed during the splicing reaction
Structure of spliceosomal proteinRNA complex: U1A binds hairpin
II of U1 snRNA
Splicing pathways
• Assembly, rearrangements, and catalysis within
the spliceosome: the splicing pathway
Early (E) complex
The A complex
Fig 13-8 steps of the spliceosomemediated splicing reaction.
The B complex
U1 snRNP replaced by U6 snRNP
Active site formed
U4 released, U2 forming RNA-RNA
hybrids with U6
Lariat initially still bound with tri-snRNP. Soon the lariat
degrades, leaving the snRNPs to be recycled.
Self-splicing introns reveal that RNA can catalyze RNA
splicing
class
abundance
mechanism
Catalytic
machinery
Nuclear premRNA
Very common;
Two
Major spliceosome
used for most
transestirification
eukaryotic genes reactions;
branch site A
Group II introns
Rare; some
Same as preeukaryotic genes mRNA
from organelles
and prokaryotes
Group I introns
Rare; nuclear
rRNA in some
eukaryotes,
organelle genes,
and a few
prokaryotic
genes
RNA enzyme encoded
by intron (ribozyme)
Two
Same as group II
transestirification
reactions;
Branch site G
Fig 13-9 group I and group II introns
Group I introns release a linear intron rather than a lariat
Fig 13-10 Proposed folding of the RNA
catalytic regions for splicing of group II
introns and pre-mRNAs.
•
Group I intron use a free G nucleoside or nucleotide.
This G species is bound by the RNA and its 3’OH
group is presented to the 5’ splice site.
•
Structure of group I intron includes
(1) a binding pocket that will accommodate any guanine
nucleoside and nucleotide.
(2) Internal guide sequence that base-pairs with the 5’
splice site sequence.
Box 13-1 group I introns can be
converted into true ribozymes.
How does the spliceosome find the splice sites reliably?
• The average exon in only some 150 nt long, whereas the
average intron is about 3,000 nt long. Thus, the exons must
be identified within a vast ocean of intronic sequences.
Fig 13-12 Errors produced by mistakes in splice-site selection.
• To avoid exon-skipping: co-transcriptional
loading of spliceosome components onto the
splice sites.(refer to 12-20)
• To avoid pseudo splice-site selection:
Fig 13-12 SR (serine-arginine rich) proteins recruit spliceosome components to
the 5’ and 3’ splice sites.
ESE: exonic splicing enhancer
A small group of introns are spliced
by an alternative spliceosome
composed of a different set of
snRNPs
Fig 13-13 The AT-AC (minor) spliceosome
catalyzed splicing.
This minor spliceosome has distinct splice site
sequences but same splicing chemistry.
Alternative Splicing
• Single genes can produce multiple products by
alternative splicing
• By alternative splicing multiple proteins can be
produced from a single gene. These different proteins
are called isoforms. They can have similar functions,
distinct functions, or even antagonistic functions.
Fig 13-14 Alternative splicing in the troponin T gene
Fig 13-15 Five ways to splice a RNA
Fig 13-16 constitutive alternative splicing: monkey virus SV40 T-antigen
5’SST: 5’ splice site used to generate the large T mRNA
5’sst: 5’ splice site used to generate the small T mRNA
Several mechanisms exist to ensure mutually
exclusive splicing
1.
Steric hindrance
2.
Combinations of major and minor splice sites
3.
Nonsense-mediated decay
The curious case of the Drosophila Dscam gene:
Mutually exclusive splicing on a grand scale
Dscam
Down syndrome cell-adhesion molecule
Alternative splicing is regulated by activators and repressors
Fig 13-22 Regulated
alternative splicing
• Proteins that regulate
splicing bind to specific
sites called exonic
(intronic) splicing
enhancers (ESE/ISE) or
silencers (ESS/ISS)
Fig 13-23 mammalian splicing repressor hnRNPI
Fig 13-25 a cascade of alternative splicing events determines the sex of a fly.
Sxl: splicing repressor
Tra: splicing activator
Exon Shuffling
• Exons are shuffled by recombination to produce genes
encoding new proteins
• Why introns are present in all organisms except bacteria???
Introns early model: due to selection pressure to speed
chromosome replication and cell division
Introns late model: due to a transposome like mechanism
Why have the introns been retained in (higher) eukaryotes??
The advantages of exon shuffling
Fig 13-26 Exons encode protein domains.
Fig 13-27 Genes made up of parts of other genes.
Fig 13-28 Accumulation, loss, and reshuffling of domains during the evolution of the
family of chromatin modifying enzymes.
Evidences of exon shuffling
1.
The boundaries between exons/introns often coincide
with boundaries between domains.
2.
Some proteins (eg. immunoglobulin) have repeating
units, which might be due to gene duplications.
3.
Related exons are sometimes found in unrelated
genes.
RNA Editing
1.
Site-specific deamination
2.
Guide RNA-directed uridine insertion or deletion
(often in trypanosomes and mitochondria).
Human apolipoprotein gene
Fig 13-29 RNA editing by deamination:
ADAR:
adenosine deaminase acting on RNA
Fig 13-26 RNA-mediated editing by guide RNA mediated U insertion
in trypanosome coxII gene.
mRNA Transport
• Once processed (capped, spliced, polyadenylated),
mRNA is packaged and exported from the nucleus into
the cytoplasm for translation
• How are RNA selection and transport achieved?
Fig 13-27 Transport of mRNAs out
of the nucleus is an active process
(export is though nuclear pore
complex with the size exclusion of
50kd)
SR protein or proteins that
recognize exon-exon boundaries
indicate a mature mRNA.
Proteins that binds to introns (eg
hnRNPs) indicate a RNA that
needs to to retained.