Download Post-Transcriptional Regulation of Gene Expression: the Many Hats

SHOWCASE ON RESEARCH
Post-Transcriptional Regulation of Gene Expression:
the Many Hats of hnRNP Proteins
Ross Smith and Jillian Dunphy
School of Molecular and Microbial Sciences, University of Queensland, QLD 4072
The cellular expression of a protein is governed not
only by the rate at which its RNA is transcribed but
also by the subsequent fate of this RNA. Alternative
splicing, nuclear export, cytoplasmic RNA trafficking,
localisation, translation and degradation all play a
significant role in determining the level and subcellular
location of many proteins. Much of our research is
focussed on RNA trafficking and splicing in
mammalian cells.
mRNA Trafficking
The development of cellular and organismal
asymmetry requires selective localisation of proteins,
including morphogenic proteins, within cells. Many
proteins possess motifs that determine where they will
ply their trade. But proteins are not the only species to
display sorting motifs; many mRNA molecules also
have elements that direct them to specific subcellular
locations, resulting in localisation of the proteins they
encode. The nature of RNA localisation pathways has
emerged primarily from studies of Xenopus and
Drosophila oocytes and embryos (1, 2). In recent years,
studies of mammalian oligodendrocytes, neurons,
fibroblasts and epithelia have added considerably to the
picture (3, 4).
Cis-Acting Elements
Selective trafficking of RNA involves the recognition of
cis-acting elements, commonly in the 3' untranslated
region (3' UTR), by cognate trans-acting proteins followed
by cytoplasmic transport in what typically appear to be
0.5-1 µm diameter granules that incorporate motor
proteins. Despite the numerous studies of RNA
localisation in Drosophila and Xenopus, two of the best
characterised RNA trafficking/localisation systems are
those involved in transport of the mRNAs encoding βactin in chicken fibroblasts and myelin basic protein
(MBP) in rat oligodendrocytes and neurons.
Two of the first mammalian RNA trafficking elements
defined were the 'zipcode' motif in the β-actin mRNA (5)
and the 21-nucleotide cis-acting sequence now termed the
heterogeneous nuclear ribonucleoprotein (hnRNP) A2
response element (A2RE), which was originally identified
by deletion analysis in MBP mRNA (6). The A2RE was
subsequently whittled down to an 11 nucleotide element
(GCCAAGGAGCC) that retains the A2RE transport
properties (7). These elements recruit their mRNAs to
specific transport granules.
Many RNA segments containing cis-acting elements
involved in trafficking have been identified, but in most
the precise location of the element is poorly defined.
From the few published 3D structures of ssDNA or
RNA in complexes with proteins, one would expect the
Vol 36 No 3 December 2005
binding sites on trans-acting factors to accommodate
8-10 nucleotides. In the A2RE these nucleotides are
contiguous, whereas in larger elements one would
anticipate that the 3D structure brings together
nucleotides separated in the linear sequence into the
binding site. This hypothesis, and the ability of
proteins to recognise even the A2RE11 cis-acting
element with a range of point mutations (8), makes
identification based solely on nucleotide sequence
difficult. Recognition of cis-acting elements at this
stage requires experimental data.
Trans-Acting Factors
A small number of RNA-binding motifs are found in
trans-acting factors, including the common RNA
recognition motif (RRM) and hnRNP K-homology
(KH) domain. From a comprehensive set of
experiments including pull-downs, cell
microinjection, biosensor and gel mobility shift assays
(7, 9, 10), we concluded that the RRM-containing
hnRNP A2 is a trans-acting factor for cytoplasmic
trafficking of RNAs possessing the A2RE11 motif.
This element is necessary and sufficient for trafficking
of MBP mRNA in oligodendrocytes and neurons, and
the A2RE11/hnRNP A2 pathway is likely to operate
in any cell with a subset of RNAs having A2RE-like
motifs. The specificity of this interaction is striking: in
the context of a kilobase RNA, mutation of a single
A2RE11 nucleotide (A8G) is sufficient to eliminate
binding to hnRNP A2 and thus RNA trafficking (Fig.
1). Oligodendrocyte treatments that reduce hnRNP
A2 levels also disrupt the transport of A2REcontaining RNA.
hnRNP A2 is ubiquitously expressed with high levels
in brain, testis and skin. Although primarily nuclear, it
is detected in the cytoplasm of many cells including
neurons, oligodendrocytes and astrocytes. It is
localised together with A2RE-containing mRNA in the
cytoplasmic transport granules. In cultured
oligodendrocytes, the levels of hnRNP A2 in the
cytoplasm correlate with the high MBP mRNA levels
seen in the most active period of process formation
(11). After this peak period, hnRNP A2 reverts to allnuclear. This may reflect changes in isoform levels
with the more cytoplasmic isoforms (lacking exon 9)
being expressed during process formation (see below).
Thus changes in alternative splicing may accompany
cellular development.
Our conclusion that hnRNP A2 is a trans-acting
factor for RNA trafficking has been strengthened by
numerous subsequent studies. Confocal microscopy
colocalisation analysis of assembling transport
granules (12) showed that hnRNP A2 is found only in
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Post-Transcriptional Regulation:
hnRNP Proteins
SHOWCASE ON
RESEARCH
Fig. 1.
(A) RNA transport in live, cultured
hippocampal neurons
microinjected with fluorescently
labelled A2RE-containing RNA
or A2RE11-containing RNA
(green) and Texas Red-labelled
dextran (red: to show cell
morphology) and observed by
confocal microscopy. The RNA
assembles into granules and
moves along the processes
(arrows in top left panel). A
single nucleotide change (A8G)
in the A2RE element of the ~200
nucleotide RNA stops its
assembly into granules, resulting
in a diffuse RNA distribution in
the soma and proximal neurites
(arrowhead in bottom left panel)
with only an occasional granule
visible in the processes. Scale
bar, 10 µm.
(B) The proportions of cells positive
for transport.
This figure is from ref 10. Copyright
2003 by the Society for Neuroscience.
granules containing MBP RNA, and not in granules
containing RNA that lacks the A2RE sequence. A2RE
binding to hnRNP A2 induces the latter to cooperatively self-associate and associate with granules.
RNAs with normally different trafficking patterns
(MBP to cell periphery; Connexin 32 to ER; GFP to
perikaryon) are segregated into different granules, but
if the A2RE is incorporated into connexin or GFP
RNAs they are incorporated into the same granules as
co-injected MBP RNA. The A2RE-mediated sorting
pathway is thus epistatic to the intrinsic sorting
pathways for GFP and connexin mRNAs.
Another trafficking pathway in mammalian cells, in
which an identified trans-acting factor binds a well defined
cognate cis-acting element, is the zipcode binding protein
(ZBP)/zipcode pathway (13, 14). ZBP1, a KH-domain
protein, binds a 54 nucleotide cis element (the 'zipcode')
that contains short tandem ZBP-binding repeats in
fibroblast β-actin RNA. A larger homologue, ZBP2, which
is enriched in the brain, also binds the actin zipcode and
has a role in localisation of β-actin mRNA. Other families
of cis-acting elements/trans-acting factors are represented
by the highly helical testis-brain RNA-binding protein (TBRBP) which binds the Y element (AGAAGCCCTATGCT),
and the cytoplasmic polyadenylation element binding
protein (CPEB), an RRM/zinc finger protein which binds
a UUUUAU sequence.
Like hnRNP A2, many of the other trans-acting
proteins involved in RNA trafficking and localisation
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are present at high concentrations in the nucleus,
where they have other functions, and many are
hnRNP-like or are hnRNP homologues.
RNA Trafficking Models
From studies of oligodendrocytes and neurons the
following picture has emerged (Fig. 2). In the nucleus,
RNAs possessing trafficking elements combine with
their cognate trans-acting factors prior to export to the
cytoplasm. In the cytoplasm, the protein-RNA complex
associates with other components of the transport
granules (including ribosomes, tRNA synthetases,
elongation factors, and other molecules needed for
translation), which then move towards the cell
periphery. The early studies of β-actin mRNA transport
showed that movement of its granules is dependent on
microfilaments in fibroblasts (14) whereas the transport
of both A2RE-containing RNA (7) and β-actin RNA
(15) in neurons is microtubule-dependent. RNA
granule movement on microtubules is governed by the
molecular motors kinesin and dynein, both of which
are present in individual granules.
Translation appears to be arrested during trafficking
and a granule protein that suppresses translation has
been identified (E. Barbarese, personal
communication). However, the mechanisms by which
the RNA-protein complexes dissociate from the
granules, and the mRNAs become anchored at their
destination and translated, are largely unknown.
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SHOWCASE ON
RESEARCH
Dendritic mRNA Transport
Post-Transcriptional Regulation:
hnRNP Proteins
In oligodendrocytes, localisation of MBP mRNA
appears to serve the function of delivering MBP
directly to nascent myelin where it is needed for
formation of compact myelin. There is no evidence
that RNAs encoding proteins other than MBP and
myelin-associated oligodendrocytic basic protein
(MOBP) are localised in these cells. By contrast, a
substantial subset of mRNAs is localised to the
dendrites in neurons, where many are translated (16).
They include mRNAs encoding microtubuleassociated protein 2 (MAP2), activity-regulated
cytoskeleton-associated protein (ARC), and the glycine
and glutamate receptors. Localised RNAs are often
found associated with ribosomes and other
components of the translation apparatus near
dendritic spines. Some of these localised RNAs, such
as that encoding the alpha-chain of Ca++/calmodulindependent kinase II (CaMKIIα) may participate in
activity-regulated protein synthesis (17). Disruption of
the 3' UTR of CaMKIIα mRNA results in its
mislocalisation (restricted to soma), a marked
reduction in post-synaptic densities, a reduction in
long-term potentiation (LTP) and impairments in
spatial memory, associative fear conditioning and
object recognition (18, 19).
Because several dendritically localised RNAs contain
A2RE-like sequences (6) and the level of hnRNP A2 in the
brain is higher than in most other cell types, interaction of
this motif with hnRNP A2 may afford a general pathway
for targeting these RNA molecules in the brain.
In addition to hnRNP A2, the proteins TB-RBP, ZBP1,
ZBP2 and MAP2-RNA trans-acting protein (MARTA)
all have established interactions with at least one
neuronal RNA element. However, it is likely that a
protein such as hnRNP A2 binds to more than one cisacting element in the same or different mRNA
molecules, and that more than one protein binds
elements within a single mRNA. For example, both
the cytoplasmic CPE and localisation elements in
CaMKIIα RNA (see above) bind proteins that influence
trafficking of this RNA in neurons, LTP and memory.
hnRNPs as Regulators of Alternative Splicing
Alternative splicing generates high protein diversity
from a limited genetic repertoire with different
isoforms potentially having distinct biological activities
or locations. Proteins belonging to the hnRNP A family
are multi-tasking; in addition to their roles in RNA
trafficking they regulate alternative splicing of RNA.
hnRNP A1, A2 and A3 isoforms have been detected
in spliceosomes at various stages of splicing (20) and
appear to regulate splice site selection by antagonising
the activities of SR (splicing factor) proteins (21). High
hnRNP levels favour exon skipping by the
spliceosome, thus affecting mRNA stability,
localisation and translatability.
hnRNP A1, A2, and A3 are both regulators of and
subject to alternative splicing. The two most abundant
of the four isoforms of hnRNP A2 appear to be solely
nuclear, but the minor forms are found at similar levels
in the nucleus and cytoplasm. We have evidence that
the minor but not the major isoforms participate in
cytoplasmic RNA trafficking (J. Hatfield, M. Landsberg
et al., unpublished data). The minor forms are
translated primarily in early development in rats, when
the need for RNA trafficking may peak (11). By
contrast, the levels of the major forms, which perform
housekeeping tasks in the nucleus, are unchanged from
birth to maturity (J. Hatfield et al., unpublished data).
Fig. 2. Model depicting trafficking of A2RE-containing RNAs.
hnRNP A2 is imported into nuclei and binds to mRNA through the A2RE cis-acting element. The A2RE
mRNA/hnRNP A2 complex is then exported to the cytoplasm. Multiple copies of the RNA-protein complex are
recruited to individual transport granules that also contain inactive translation machinery. The transport granules
attach to microtubules and are transported to the site of translation (in this case to dendritic spines). At its
destination the mRNA is anchored and translated. Each of the steps in this process, including RNA degradation,
is a potential post-transcriptional control point for gene expression.
Vol 36 No 3 December 2005
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Post-Transcriptional Regulation:
hnRNP Proteins
SHOWCASE ON
RESEARCH
Overexpression of splice variants hnRNP A2 and B1
and an increase in hnRNP B1 relative to A2 are
observed in lung carcinogenesis and recapitulate the
changes during foetal lung development. This
reinforces the view that hnRNP A2/B1 is an oncodevelopmental protein and suggests a role for the
alternative splicing of this protein in the regulation of
tumourigenesis (22).
Conclusions and Perspectives
The multi-tasking hnRNP A/B family of proteins
play important roles in cytoplasmic mRNA trafficking.
Two prime needs for advancement of our
understanding of RNA trafficking are the precise
identification of further cis-acting elements and their
trans-acting factors, and elucidation of the 3D
structures of their complexes. Such information will
define better the principles of recognition of RNA
elements by proteins.
We have focussed here on hnRNP A2, but this protein
has two paralogues, hnRNP A1 and A3, and we have
evidence that the latter is also involved in RNA
transport. It will be interesting to define more fully the
connections between these three proteins; although they
have partially overlapping functions they also show
some clear differences. For example, their levels change
differently during the cell cycle, they have different
effects on cell growth (23), and they are subject to
different patterns of Arg dimethylation in their RGG
boxes. It seems that cells do not simply produce the
three paralogues as a molecular insurance policy.
Additionally, these hnRNP paralogues have nuclear
functions that include the regulation of pre-mRNA
splicing and all have multiple splice variants that may
differ in localisation and function. The possibility that
the four isoforms of hnRNP A2 fulfil different functions
warrants further investigation, both to improve our
understanding of this protein and to clarify the general
role played by alternative splicing, which is emerging
as a critical step in the regulation of gene expression.
hnRNP A2 should prove useful in investigating the
roles of different isoforms; it is relatively abundant and
there is evidence for some of its isoforms being
confined to separate subcellular compartments.
The dual roles of hnRNP A proteins in mRNA
splicing and trafficking highlight the co-ordination of
RNA processing and localisation. The importance of
appropriate RNA processing and trafficking to the
quantity, quality and localisation of proteins is readily
apparent for both A2RE-containing RNAs and hnRNP
A proteins themselves.
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