V5: mRNA translation

SS 2009 – lecture 5Biological Sequence Analysis

1

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


2

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.

www.wikipedia.org


3

www.wikipedia.org

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.

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.

processing: 5‘ cap addition

Calero et al. Nat. Struct. Biol. 9, 912 (2002)

m7GpppG binding to nuclear cap-binding protein complex


4

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

processing: editing


5

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.

www.wikipedia.org


6

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.

www.wikipedia.org

RNA splicing

www.wikipedia.org

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).


8

splicing repression

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 ribonucleo-

proteins (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein

(PTB).

www.wikipedia.org

There are 2 major types of

RNA sequence elements

present in pre-mRNAs, and

specific RNA-binding

proteins bind to each of

these elements.


9

splicing activation

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.

www.wikipedia.org

Splicing Enhancers are

sites to which splicing

activator proteins bind,

increasing the probability

that a nearby site will be

used as a splice junction.


10

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


11

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


12

Example: alternative splicing of Drosophila dsx pre-mRNA

De-Leon, Annu Rev. Biophys Biomol Struct. 36, 191 (2007)

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 trans-

criptional 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.


13

importance of alternative splicing

Alternative splicing is of great importance to genetics - it invalidates the old "one-

gene-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.



14

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.


Arniges et al. J. Biol. Chem. 281, 1580 (2006)

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.

Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)

Identification of TRMP3 splice variants from mouse brain

A, schematic diagram of the mouse Trpm3 gene, comprising 28 exons.


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.


Pore regions of splice variants

D, putative pore regions of TRPM31 and TRPM32 compared with the

corresponding mouse sequences of TRPM6, TRPM7, TRPV5, and TRPV6.

The 12 additional amino acid residues present in TRPM31 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.


TRPM3 functions as cation channel

Heterologous expression of TRPM31 induces outwardly rectifying cation

currents inhibited by intracellular Mg2+.

A,current-voltage relationship of a TRPM31-expressing cell in standard Ringer

or NMDG solution within 60 s after establishing the whole cell patch clamp

configuration.


Permeability for divalent cations

TRPM31 and TRPM32 display large differences in their relative

permeability ratios for divalent cations.

A, comparison of TRPM31 and TRPM32 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.


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).


Effect of extracellular cations

Inhibition of TRPM3-dependent currents

by extracellular cations.

A, comparison of TRPM31 and TRPM32

currents at 80 mV and 80 mV in extra-

cellular solutions containing indicated

amounts of Na+.

Outward currents through TRPM31 are

unaffected by extracellular Na+, whereas

outward currents through TRPM32 are

inhibited in a dose-dependent manner by

these ions.

B, statistical analysis of recordings with

varying concentrations of Na+, K+, Ca2+, and

Mg2+.

TRPM32 is inhibited byall cations tested on the extra-cellular side.


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.


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, TRPM31, preferentially conducts monovalent cation influx,

whereas TRPM32 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.


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 (TRPM32) and 20

(TRPM31) 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 TRPM31 compared with

TRPM32, perhaps simply because of increased electrostatic repulsion.

Block of TRPM3 Channels by Intra- and Extracellular Cations — Both TRPM31

and TRPM32 are regulated by physiological concentrations of intracellular Mg2+,

similar to related members of the TRPM family such as TRPM6 and TRPM7.


26

end of translation: action of ribosome

www.wikipedia.org


27

additional slides


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.


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.


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.


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.


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 1M

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.


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.


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 TRPV4-

D variants.


Heteromerization of TRPV4 variants

B, FRET efficiencies

determined between different

TRPV4 variants showed

heterooligomerization only for A

and D proteins.


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.


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.


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.


39

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.



40

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.



41

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)


V5: mRNA translation

Documents

Transcript of V5: mRNA translation