Transcript ppt - Chair of Computational Biology

V5: mRNA translation
The brief existence of an mRNA
molecule begins with transcription
and ultimately ends in degradation.
During its life, an mRNA molecule
may also be processed, edited, and
transported prior to translation.
Eukaryotic mRNA molecules often
require extensive processing and
transport, while prokaryotic
molecules do not.
www.wikipedia.org
Biological Sequence Analysis
SS 2009 – lecture 5
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eukaryotic pre-mRNA processing
The short-lived, unprocessed or partially processed, product of transcription by
RNA polymerase is termed pre-mRNA.
Once completely processed, it is termed mature mRNA.
Processing of mRNA differs greatly among eukaryotes, bacteria and archea.
Non-eukaryotic mRNA is essentially mature upon transcription and requires no
processing, except in rare cases.
Eukaryotic pre-mRNA, however, requires extensive processing.
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Biological Sequence Analysis
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processing: 5‘ cap addition
A 5' cap is a modified guanine nucleotide that has been added to the 5' end of a
eukaryotic messenger RNA shortly after the start of transcription.
The 5' cap consists of a terminal 7-methylguanosine residue which is linked
through a 5'-5'-triphosphate bond to the first transcribed nucleotide.
Its presence is critical for recognition by the ribosome,
pre-mRNA splicing, 3‘end formation, U snRNA
transport, and regulation of decay.
Cap addition is coupled to transcription, and
occurs co-transcriptionally, such that each
influences the other.
Shortly after the start of transcription, the 5' end
of the mRNA being synthesized is bound by a
cap-synthesizing complex associated with RNA
polymerase.
www.wikipedia.org
m7GpppG binding to nuclear cap-binding protein complex
Biological Sequence Analysis
SS 2009 – lecture 5
Calero et al. Nat. Struct. Biol. 9, 912 (2002)
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processing: editing
In some instances, an mRNA will
be edited, changing the
nucleotide composition of
that mRNA.
mRNA has been observed in tRNA,
rRNA, and mRNA molecules of eukaryotes but not prokaryotes.
RNA editing mechanisms include nucleoside modifications such as C to U and A
to I deaminations, as well as non-templated nucleotide additions and insertions.
RNA editing alters the amino acid sequence of the encoded protein so that its
sequence differs from that predicted from the genomic DNA sequence.
An example in humans is the apolipoprotein B mRNA, which is edited in some
tissues, but not others. Here, the editing creates an early stop codon, which upon
translation, produces a shorter protein.
www.wikipedia.org
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processing: polyadenylation
Polyadenylation is the covalent linkage of a polyadenylyl moiety to an mRNA
molecule. In eukaryotic organisms, most mRNA molecules are polyadenylated at
the 3' end.
The poly(A) tail and the protein bound to it aid in protecting mRNA from
degradation by exonucleases.
Polyadenylation is also important for
- transcription termination,
- export of the mRNA from the nucleus, and
- translation.
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Biological Sequence Analysis
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processing: polyadenylation
Polyadenylation occurs during and immediately after transcription of DNA into RNA.
After transcription has been terminated, the mRNA chain is cleaved through the
action of an endonuclease complex associated with RNA polymerase.
After the mRNA has been cleaved, around 250 adenosine residues are added to
the free 3' end at the cleavage site.
This reaction is catalyzed by polyadenylate polymerase.
Just as in alternative splicing, there can be more than one polyadenylation variant
of a mRNA.
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Biological Sequence Analysis
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RNA splicing
Splicing is the process by which pre-mRNA is modified to remove certain
stretches of non-coding sequences called introns; the stretches that remain
include protein-coding sequences and are called exons.
This is needed for the typical eukaryotic messenger RNA before it can be used
to produce a correct protein through translation.
For many eukaryotic introns, splicing is done in a series of reactions which are
catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins
(snRNPs), but some RNA molecules are also capable of catalyzing their own
splicing (ribozymes).
www.wikipedia.org
splicing repression
There are 2 major types of
RNA sequence elements
present in pre-mRNAs, and
specific RNA-binding
proteins bind to each of
these elements.
Silencers are sites to which splicing repressor proteins bind, reducing the
probability that a nearby site will be used as a splice junction.
These can be located in the intron itself (intronic splicing silencer, ISS) or in a
neighboring exon (exonic splicing silencer, ESS). They vary in sequence, as well
as in the types of proteins that bind to them.
The majority of the repressors that bind are heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein
(PTB).
www.wikipedia.org
Biological Sequence Analysis
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splicing activation
Splicing Enhancers are
sites to which splicing
activator proteins bind,
increasing the probability
that a nearby site will be
used as a splice junction.
Splicing enhancers also may occur in the intron (intronic splicing enhancer, ISE)
or exon (exonic splicing enhancer, ESE).
Most of the activator proteins that bind to ISEs and ESEs are members of the SR
protein family. Such proteins contain RNA recognition motifs and arginine and
serine-rich (RS) domains.
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Biological Sequence Analysis
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alternative splicing (AS)
Alternative splicing is a RNA splicing variation mechanism in which the exons of
the primary gene transcript, the pre-mRNA, are separated and reconnected so as
to produce alternative ribonucleotide arrangements.
These linear combinations are then translated into different proteins.
In this way, AS uses genetic expression to facilitate the synthesis of a greater
variety of proteins.
www.wikipedia.org
Biological Sequence Analysis
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5 basic modes of alternative splicing
(1) Exon skipping: here, an exon may be spliced out
of the primary transcript or retained. This is generally
the most common mode in mammalian pre-mRNAs.
(2) Mutually exclusive exons: One of two exons is
retained in mRNAs after splicing, but not both.
(3) Alternative donor site: An alternative 5' splice
junction (donor site) is used, changing the 3' boundary
of the upstream exon.
(4) Alternative acceptor site: An alternative 3' splice
junction (acceptor site) is used, changing the 5'
boundary of the downstream exon.
(5) Intron retention: A sequence may be spliced out as
an intron or simply retained. This is distinguished from
exon skipping because the retained sequence is not
flanked by introns. If the retained intron is in the
coding region, the intron must encode amino acids in
frame with the neighboring exons, or a stop codon or
a shift in the reading frame will cause the protein to be
non-functional. This is generally the rarest mode in
mammals.
www.wikipedia.org
SS 2009 – lecture 5
Biological Sequence Analysis
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Example: alternative splicing of Drosophila dsx pre-mRNA
Pre-mRNAs from the D. melanogaster gene
dsx contain 6 exons.
In males, exons 1,2,3,5,and 6 are joined to
form the mRNA, which encodes a transcriptional regulatory protein required for
male development.
In females, exons 1,2,3, and 4 are joined,
and a poly-A signal in exon 4 causes
cleavage of the mRNA at that point. The
resulting mRNA is a transcriptional
regulatory protein required for female
development.
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
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importance of alternative splicing
Alternative splicing is of great importance to genetics - it invalidates the old "onegene-one-protein" hypothesis.
External information is needed in order to decide which polypeptide is produced,
given a DNA sequence and pre-mRNA.
Possibly, this was a very important step towards higher efficiency of eukaryotic
genomes, because information can be stored much more economically.
Several proteins can be encoded in a DNA sequence whose length would only be
enough for two proteins in the prokaryote way of coding.
Alternatively, a new protein can evolve without changing the DNA of a gene.
Instead, the same effect can be achieved by differential regulation.
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
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importance of alternative splicing
Humans have only about twice as many genes as Caenorhabditis elegans or the
fly Drosophila melanogaster.
How can one explain the greater complexity of humans?
Hypothesis: The greater complexity of humans, or vertebrates generally, might be
due to higher rates of AS in humans than in invertebrates.
However, EST studies showed that the frequency of AS is similar in human to that
in mouse, rat, cow, fly, worm, and the plant Arabidopsis thaliana.
The "record-holder" for AS is a D. melanogaster gene called Dscam, which has
38,016 splice variants.
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
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Example of AS: Transient Receptor Potential channels
The general topology of a TRP subunit consists of
- 6 predicted TM domains with
- a putative pore loop between TMD5 and TMD6 and
- intracellular N- and C-terminal regions of variable length,
the former containing multiple ankyrin (ANK) repeats in the TRPC, TRPA,
TRPN, and TRPV subfamilies.
Functional TRP channels are supposed to result following the assembly of 4 TRP
subunits.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Identification of TRMP3 splice variants from mouse brain
A, schematic diagram of the mouse Trpm3 gene, comprising 28 exons.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
TRPM3 splice variants
C, schematic presentation of TRPM3 with transmembrane domains 1–6, coiled coil
region (cc), and TRP homology domain (Trp).
Novel mouse TRPM3 protein variants shown as thick black lines are compared with
the human variants hTRPM3a–f and hTRPM31325. The numbers of amino acid
residues of each variant are indicated in parentheses.
Starting from residue 156, mouse and human TRPM3 have 97% sequence identity.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Pore regions of splice variants
D, putative pore regions of TRPM31 and TRPM32 compared with the
corresponding mouse sequences of TRPM6, TRPM7, TRPV5, and TRPV6.
The 12 additional amino acid residues present in TRPM31 are indicated.
Identical residues are boxed in black, conserved in gray.
An Asp residue that determines Ca2+ permeation of the TRPV5/TRPV6 pore is
marked by an asterisk. Residues proposed to build the selectivity filter of
TRPV6 are underlined.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
TRPM3 functions as cation channel
Heterologous expression of TRPM31 induces outwardly rectifying cation
currents inhibited by intracellular Mg2+.
A,current-voltage relationship of a TRPM31-expressing cell in standard Ringer
or NMDG solution within 60 s after establishing the whole cell patch clamp
configuration.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Permeability for divalent cations
TRPM31 and TRPM32 display large differences in their relative
permeability ratios for divalent cations.
A, comparison of TRPM31 and TRPM32 currents at 80 mV and 80 mV in
extracellular solutions containing indicated amounts of Ca2+.
B, reversal potential during the experiment shown in panel A.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Identification of TRMP3 variants from mouse brain
C and D, statistical analysis of reversal
potential measurements in experiments
similar to that shown in panel B during the
application of solutions containing the
indicated concentration of Ca2+ (C) or
Mg2+ (D) as the only permeable ion.
Continuous thin lines show the expected
reversal potential calculated from
Goldman-Hodgkin-Katz theory for the
indicated relative permeability ratios.
Each point represents the mean of 3–15
independent measurements (at a divalent
concentration of 10 mM p < 0.001,
otherwise at least p < 0.05).
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Effect of extracellular cations
Inhibition of TRPM3-dependent currents
by extracellular cations.
A, comparison of TRPM31 and TRPM32
currents at 80 mV and 80 mV in extracellular solutions containing indicated
amounts of Na+.
Outward currents through TRPM31 are
unaffected by extracellular Na+, whereas
outward currents through TRPM32 are
inhibited in a dose-dependent manner by
these ions.
B, statistical analysis of recordings with
varying concentrations of Na+, K+, Ca2+, and
Mg2+.
TRPM32 is inhibited by
all cations tested on the extracellular side.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Summary
Alternative Splicing Switches the Ion Selectivity of TRPM3 Channels—
The selectivity of ion channels is thought to be determined by the geometry and
charge distribution of the selectivity filter, usually envisioned as the narrowest
part of the channel pore.
Typically, all members of an ion channel family, such as voltage-gated Na+, K+, or
Ca2+ channels, share common ionic selectivities.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Summary II
The TRP family of ion channels is unusual in this respect as its members have
quite diverging cationic selectivity profiles.
The Trpm3 gene adds extra complexity to this picture, because two channels can
be expressed from this gene with entirely different ionic selectivities.
One channel, TRPM31, preferentially conducts monovalent cation influx, whereas
TRPM32 strongly favors divalent entry.
In vivo, such a change in ionic selectivity must be expected to have considerable
consequences for the function of the channel and the physiology of the cell that
expresses it.
The switch of ionic selectivity in TRPM3 variants is due to removal of a short
stretch of 12 amino acid residues and exchanging 1 further residue within the
linker domain between the presumed fifth and sixth transmembrane regions.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
Summary III
Compared with the presumed pore regions of other members of the TRP family,
the pore loop of TRPM3 is considerably longer by 8 (TRPM32) and 20 (TRPM31)
additional amino acid residues.
The domains that build the proposed selectivity filter of the Ca2+-selective
TRPV5/V6 channels are conserved in TRPM3 proteins.
The splicing within the TRPM3 channel pore introduces additional, positively
charged amino acid residues into this domain.
This might decrease the Ca2+ permeability of TRPM31 compared with TRPM32,
perhaps simply because of increased electrostatic repulsion.
Block of TRPM3 Channels by Intra- and Extracellular Cations — Both TRPM31
and TRPM32 are regulated by physiological concentrations of intracellular Mg2+,
similar to related members of the TRPM family such as TRPM6 and TRPM7.
Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)
end of translation: action of ribosome
www.wikipedia.org
Biological Sequence Analysis
SS 2009 – lecture 5
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additional slides
Biological Sequence Analysis
SS 2009 – lecture 5
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TRPV4 channels
The non-selective cation channel TRPV4 is a member of the transient
receptor potential (TRP) family of channels.
TRPV4 shows multiple modes of activation and regulatory sites, enabling it to
respond to various stimuli, including
osmotic cell swelling,
mechanical stress,
heat,
acidic pH,
endogenous ligands,
high viscous solutions, and
synthetic agonists such as 4-phorbol 12,13-didecanoate.
TRPV4 mRNA is expressed in a broad range of tissues, although functional tests
have only been carried out in a few:
endothelial, epithelial, smooth muscle, keratinocytes, and DRG neurons.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Cloning of TRPV4 variants from human airway epithelial cells
A reverse transcriptase-PCR-based cloning process identified 5 variants of the
TRPV4 channel in human tracheal epithelial cells.
2 of the cloned cDNAs corresponded to the already described
- TRPV4 isoform A (fulllength cDNA) and
- TRPV4 isoform B (lacking exon number 7, 384–444 amino acids).
We also identified 3 new splice variants affecting the cytoplasmic N-terminal region.
TRPV4-C lacks exon 5 (237–284 amino acids),
TRPV4-D presents a short deletion inside exon 2 (27–61 amino acids), and
TRPV4-E (237–284 and 384–444 amino acids) is produced by a double alternative
splicing lacking exons 5 and 7.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Different splice variants of TRPV4
A, schematic diagram showing the
intracellular N-terminal region of
the human TRPV4 channel
(amino acids 1–471).
Exons and the corresponding
amino acids lost in each TRPV4
isoform are indicated by numbers.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Functional analysis of TRPV4 variants: intracellular [Ca2+]
The TRPV4-A channel responds to
a wide variety of stimuli.
Here, HeLa cells were transiently
transfected and intracellular Calcium
concentration was determined via
Fura-2 ratios as reponse to 3 well
known activators of TRPV4-A: 30%
hypotonic solution, 1 M 4-PDD, or
10 M arachidonic acid
 Only TRPV4-A and TRPV4-D
show channel activity.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
TRPV4-A and D produce functional channels
TRPV4-A and TRPV4-D isoforms
produce functional channels with
similar properties when expressed
in HEK-293 cells.
A, current traces obtained from
TRPV4-A and TRPV4-D-expressing
HEK-293 cells at the indicated
voltages in the presence of 1M
4-PDD. Dashed lines indicate the zero
current level.
B, I–V relationship of 4-PDD-activated
TRPV4-A (open circle) and TRPV4-D
(closed circle) channels in inside-out
patches.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Retention in ER
Co-localization experiments (not shown):
TRPV4-B, C and E are trapped in the ER and not translocated to the plasma
membrane.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Homomerization of TRPV4 variants
FRET efficiencies determined between
identical CFP- and YFP-fused TRPV4
variants (A–E) transiently cotransfected
in HEK-293 cells.
High FRET efficiencies corresponding to
homomultimer formation could only be
demonstrated for TRPV4-A and TRPV4D variants.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Heteromerization of TRPV4 variants
B, FRET efficiencies
determined between different
TRPV4 variants showed
heterooligomerization only for A
and D proteins.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Summary
This study of oligomerization, localization, and channel activity of human TRPV4
splice variant identified the N-terminal ANK repeats as key molecular
determinants of subunit assembly and subsequent processing of the assembled
channel.
Five TRPV4 variants (TRPV4-A–E) cloned from human airway epithelial cells
were grouped into two classes:
group I: TRPV4-A and TRPV4-D
group II: TRPV4-B, TRPV4-C, and TRPV4-E.
Group I variants are correctly processed and targeted to the plasma membrane
where they form functional channels with similar electrophysiological properties.
Variants from group II, which are lacking parts of the ANK domains are unable to
oligomerize and were retained intracellularly, in the ER.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Summary II
Discovery of three important traits of TRPV4 biogenesis.
1) Glycosylation of TRPV4 channel involves ER to Golgi transport with the
corresponding change in the N-linked oligosaccharides from the high mannose type
characteristic of the ER to the complex type characteristic of the Golgi apparatus,
without apparent O-glycosylation.
2) TRPV4-A subunits oligomerize in the ER.
3) Impaired subunit assembly of type II variants is because of the lack of N-terminal
ANK domains and causes protein retention in the ER.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
Summary III
Ion channel functional diversity is greatly enlarged by both the presence
of splice variants and heteromerization of different pore-forming and regulatory
subunits. Alternative splicing is a major contributor to protein diversity.
Within the TRP family of ion channels several splice variants have been identified,
some of them resulting in lack of responses to typical stimuli, others modifying the
pore properties, and those exerting dominant negative effects.
Group II TRPV4 splice variants have been identified in two unrelated, human airway
epithelial cell lines. Considering the relevance of TRPV4 channels in epithelial
physiology, a change in the expressed ratio of group I to group II variants, favoring
the later, may modify normal epithelial functioning.
Splicing can be regulated by several stressing stimuli including pH, osmotic, and
temperature shocks, all of them being also activating stimuli of the TRPV4.
Arniges et al. J. Biol. Chem. 281, 1580 (2006)
mRNA translation
In activation, the correct amino acid is covalently bonded to the correct transfer
RNA (tRNA). While this is not technically a step in translation, it is required for
translation to proceed. The amino acid is joined by its carboxyl group to the 3' OH
of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, it is
termed "charged".
Initiation involves the small subunit of the ribosome binding to 5' end of mRNA
with the help of initiation factors (IF). Termination of the polypeptide happens
when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA). When
this happens, no tRNA can recognize it, but a releasing factor can recognize
nonsense codons and causes the release of the polypeptide chain. The 5' end of
the mRNA gives rise to the proteins N-terminal and the direction of translation can
therefore be stated as N->C.
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
SS 2009 – lecture 5
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mRNA translation
The mRNA carries genetic information encoded as a ribonucleotide sequence
from the chromosomes to the ribosomes. The ribonucleotides are "read" by
translational machinery in a sequence of nucleotide triplets called codons. Each
of those triplets codes for a specific amino acid.
The ribosome and tRNA molecules translate this code to a specific sequence of
amino acids. The ribosome is a multisubunit structure containing rRNA and
proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs
are small noncoding RNA chains (74-93 nucleotides) that transport amino acids to
the ribosome. tRNAs have a site for amino acid attachment, and a site called an
anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet
that codes for their cargo amino acid.
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
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mRNA translation
Aminoacyl tRNA synthetase catalyzes the bonding between specific tRNAs and
the amino acids that their anticodons sequences call for.
The product of this reaction is an aminoacyl-tRNA molecule. This aminoacyl-tRNA
travels inside the ribosome, where mRNA codons are matched through
complementary base pairing to specific tRNA anticodons. The amino acids that
the tRNAs carry are then used to assemble a protein. The energy required for
translation of proteins is significant. For a protein containing n amino acids, the
number of high-energy Phosphate bonds required to translate it is 4n-1.
The rate of translation varies; it is significantly higher in prokaryotic cells (up to
17-21 amino acid residues per second) than in eukaryotic cells (up to 6-7 amino
acid residues per second)
De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)
Biological Sequence Analysis
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