11-2-11 RNA Splicing and Protein Synthesis
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Processing of ribosomal and transfer RNAs
mRNA modification and splicing
Catalytic functions of RNA
The genetic code
Amino acid activation
Ribosome structure
Protein synthesis
a. initiation, elongation and termination
b. inhibition of protein synthesis
8. Secretory and membrane proteins
9. Regulation of protein synthesis
Virtually all initial transcription products are
processed in eukaryotes
Eukaryotic ribosomal RNAs are generated by
cleavage of a precursor molecule
Nucleolar RNA polymerase I transcribes a single
45S precursor containing 18S, 28S and 5.8S
rRNAs
18S rRNA – component of small 40S subunit
28S, 5.8S rRNAs – components of large 60S
subunit
The 5S component of the 60S subunit is
transcribed by RNA polymerase III
Nucleotides in the pre-rRNA are extensively
modified prior to cleavage
Modification of pre-rRNA bases and ribose is
catalyzed by snoRNPs (small nucleolar
ribonucleoproteins) consisting of snoRNA and
several proteins
Cleavage and additional modification of pre-rRNA
leads to production of mature rRNAs that are
assembled together with ribosomal proteins
into eukaryotic ribosomes
Virtually all steps occur in the nucleolus
RNA Polymerase III transcribes Transfer
RNAs that are then extensively processed
5’ nucleotides (the 5’ leader) are cleaved by
RNase P
CCA, the amino acid attachment site, is added to
the 3’ end by CCA adding enzyme
tRNA bases and riboses are extensively modified
Some pre-tRNAs contain introns that must be
removed by splicing by endonuclease and
ligase
Messenger RNAs are modified and spliced
RNA polymerase II transcription products are
extensively modified
The 5’ end of pre-mRNA is modified by addition
of a 5’-5’ cap consisting of 7-methylguanylate
(cap 0)
Adjacent ribose residues may be methylated to
form cap 1 or cap 2
5’ Caps stabilize mRNAs and enhance translation
Most pre-mRNA 3’ ends are modified by
polyadenylation to create poly(A) tails
3’ nucleotides are removed from the primary
transcript before addition of poly (A)
An internal AAUAAA sequence in the primary
transcript is recognized by a specific
endonuclease that removes downstream nt’s
Poly(A) polymerase then adds about 250 adenylate
residues to the 3’ end of the transcript
Poly(A) tails stabilize the transcript and enhance
translation efficiency
Introns are spliced from pre-mRNAs
Introns are precisely marked by splice sites
Introns begin with GU and end with AG
5’ splice sites are marked by the consensus
sequence AGGUAAGU in vertebrates
3’ splice sites are marked by the polypyrimidine
tract (10 U or C residues)
Small nuclear RNAs in spliceosomes catalyze
pre-mRNA splicing
snRNAs contain fewer than 300 nucleotides and
some are essential to the splicing process
snRNAs associate with specific proteins to form
small nuclear ribonucleoprotein particles
(snRNPs), or “snurps”
In mammals splicing is initiated by recognition of
the 5’ splice site by the U1 snRNP, which
contains a 6 base pair sequence that base
pairs with the 5’ splice site
U1 snRNP binding initiates spliceosome
assembly
U2 snRNP then binds the “Branch site”
Preassembled U4-U5-U6 join U1-U2 to
complete spliceosome assembly
Splicing begins when U5 interacts with
the exon sequence in the 5’ splice
site and then the 3’ exon
U6 disengages from U4 and interacts
with U2 and the 5’ end of the intron
displacing U1
U2 and U6 thus form the catalytic center
U4 is an inhibitor that masks U6
until the specific splice sites
are aligned
The ends of the intron are thus
brought together, resulting in
“transesterification”
The 5’ end of the intron is cleaved to
produce a lariat intermediate
with the first G of the intron
linked to the A in the branch
region
U5 holds the 3’ end of exon 1 near
the 5’ end of exon 2, resulting
in transesterification 2
Transesterification 2 connects exon 1 with exon 2,
generating the spliced product
U2, U5 and U6 bound to the excised lariat intron are
released to complete the splicing reaction
ATP powered RNA helicases are required to unwind
RNA helices and create the alternative base pairs
needed in splicing
Mutations that affect Pre-mRNA splicing
cause disease
Mutations can be cis-acting (affecting pre-mRNA)
or trans-acting (affecting splicing factors)
Cis-acting mutations cause some thalassemias
hereditary anemia caused by defective
hemoglobin synthesis
The hemoglobin b gene has 3 exons and 2 introns
Cis-acting mutations can affect splice sites
Splicing mutations result in incorrectly spliced
mRNA that create translation stop sites
preventing formation of full length hemoglobin b
Mutations affecting splicing are estimated to cause
at least 15% of all genetic diseases
Alternative splicing yields protein diversity
Different combinations of exons within the same
gene may be spliced into mature RNA to
produce distinct forms of the protein for
specific tissues, developmental stages or
signaling pathways
Alternative splicing is controlled by trans-acting
factors that differ in different cells
Alternative splicing expands the versatility of
genomes via combinatorial control
In humans two different hormones are produced
from a single calcitonin-CGRP pre-mRNA
calcitonin-generelated protein,
a vasodialator
calcium and
phosphate
metabolism
RNA can function as a catalyst - Ribozymes
Splicing is mainly catalyzed by RNA molecules,
with proteins playing a supporting role
RNase P has an RNA component that contributes
to cleaving nucleotides from the 5’ end of
tRNA precursors
Ribosomal RNAs are catalytic during translation
Ribosomal RNA processing in Tetrahymena
contains a 414 bp “self-splicing” intron