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properties of RNA-binding by the Pumilio–FBF (PUF) protein family.

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Pumilio–FBF (PUF) proteins are a relatively small family of
RNA binding proteins, with two members in mammals, one in Drosophila, six in yeast and more than eight in C. Elegans. They are mainly involved in
controlling gene expression, but also take part in ribosome biogenesis and
bind non-coding RNAs. Because their RNA-binding domain, the conserved PUF
homology domain (PUF-HD), can be fused to various effectors and designed to
specifically recognise diverse RNA sequence, PUFs are becoming valuable tools
in the research and therapeutic fields. Thus, a good understanding of PUF RNA-binding
is of great importance and factors affecting the PUFs’ affinity and specificity
will be explored in this review.

PUF structure
and function

PUF proteins are involved in a variety of processes, such as
embryogenesis, cell fate, receptor signalling pathways, and neurone functioning.
They bind a Pumilio Recognition Element (PRE) (termed differently for different
PUFs), which is most commonly found in the 3’UTR of messenger RNAs and
generally act as a scaffold for the binding of downstream regulators. This
binding usually results in a decrease in protein expression, but can sometimes promote
translation or affect mRNA localisation. Downregulation is predominantly
achieved by recruiting the deadenylase complex and subsequent mRNA degradation.
However, PUF binding can also reveal miRNA binding sites, causing the mRNA to
associate with the RISC complex, or affect the translation phase.

The canonical
PUF-HD is crescent shaped and comprises eight structurally similar
repeats, each made up of 3 ?-helices and 1′ and 8′ pseudo-repeats
with 1-2 ?-helices.
Three amino acids on the concave-side ?-helix of each repeat, the tripartite recognition
motif (TRM), interact with one RNA base allowing the PUF-HD to specifically
bind 8nt sequences (fig.1). Two of these residues form bonds with the base edge-on,
while the third stacks between bases. The more acidic convex surface of the
PUF-HD is bound by other proteins. Other regions of the protein, differ with
the PUF and are either unstructured or their structures still require



PUF proteins have multiple established and suggested uses in
research and therapeutics, which usually involve fusion of the PUF-HD to an
effector domain, such as a fluorescent protein, endonuclease, polyadenylation
or splicing factor. The most frequently mentioned applications are RNA imaging in vivo and control of protein
expression, for which PUF-HD fusion proteins are especially valuable as they can
be utilised to both downregulate and upregulate protein synthesis. A
particularly valuable feature of PUFs, especially when constructing regulatory
networks or cascades, is the ability to control mRNA translation, exerting a
faster effect on gene expression. For example, if PREs are inserted in front of
the start codon, the PUF-HD can sterically hinder the progression of the
ribosome – a system which works in both eukaryotes and prokaryotes and even
allows specific repression of individual genes within a polycistronic mRNA. Because
the PUF-HD can be mutated or even constructed de novo from repeat-like modules to recognise a variety
of sequences, it can also be used to bind endogenous genes. Thus, PUF
constructs were suggested for use in gene therapy. For example, the PUF-HD can
be made to recognise repeating sequences, such as the (CUG)n repeats
responsible for type 1 myotonic dystrophy and could potentially compete with
the splicing factors sequestered by them. Diseases caused by improper splicing,
such as cancer associated with certain splicing isoforms of the VEGF-A gene, may
also be treated by PUF constructs. It was shown possible for a PUF-HD – splicing
factor fusion to target the gene of this endogenous antitumor agent and restore
the normal splicing balance. Novel PUF-HD-like proteins can now be designed to
minimise off-target effects, which have previously been a concern, by targeting
longer 16-18 nt sequences. Importantly, it is possible to modulate the extent
of any effect exerted by PUF binding by varying the number of PREs or by
manipulating protein affinity.


affecting PUF affinity and specificity


PUF proteins usually
bind an 8nt 5’UGU…AU’3 motif with high affinity and with high specificity at
the edges of the sequence, while more ambiguity is allowed for the
middle nucleotides. The
nucleotides bound are primarily determined by the TRMs. All three amino acids
contribute to the affinity, while specificity is mostly determined by the edge-on
residues as they form specific interactions with each base (fig.1C). By mutating
these, it is possible to confer recognition of any base to any repeat. While
the stacking residues do not dictate the specificity of the repeat, they have
been shown to broaden or restrict it, an effect which can also be achieved
through mutation. Importantly, unlike the edge-on residues, they can also
influence the specificity of the neighbouring base. The picture, however is not
as simple as a “one TRM specifies one base” model, as it has been shown that
RNA can be bound in alternate modes. A base which interacts with a TRM through
its Watson-Crick edge can bind the same TRM of a slightly different PUF-HD with
its Hoogsteen edge. Alternatively, a single TRM can, in some cases, tolerate
more than one base. Further complications arise as bases are sometimes omitted,
especially in the middle of the motif, when certain sequences are bound. Thus,
when designing novel PUFs, specificity is sometimes difficult to achieve. The
mechanism by which different binding modes are achieved remains uncertain:
other factors beyond the TRM are likely involved in PUF specificity.


One factor which can affect specificity is the curvature of
the PUM-HD. It has been noticed that some PUF proteins, such as Puf-11, FBF-2 (C. elegans) and Puf5p (S. cerevisiae) are able to bind PREs
longer than 8nt despite having eight repeats. It appears that their flatter
curvatures allow the PUF-HDs to bind longer RNA sequences by omitting some of
the nucleotides in the middle of their PREs. For example, FBF-2, which has a
flatter angle between repeats 4 and 5, binds 9nt sequences and Puf5p
orthologs, having some of the flattest curvatures among known PUFs, also bind
the longest PREs (8-12nt). Because of the reduced specificity in the middle of
the PRE, flatter PUF proteins might produce more off target effects – an
important point to consider when designing new PUFs, especially when constructing
them de novo. A technique has been
recently proposed for constructing proteins with desired curvatures by using
repeating block and junction modules which could potentially be
applicable to PUF design and is yet to be tested.

Binding partners

factor which can affect the affinity and specificity of Puf proteins is
interaction with binding partners. One such partner is the Nanos (Nos) protein,
a Zinc-finger RBP long known to interact with Pumilio in Drosophila. In the presence of Pumilio, it binds a Nanos Response
Element (NRE), which includes the PRE, increases the number of genes regulated
by Pumilio and enhances mRNA repression. It does so by binding both the protein
and mRNA like a clamp, stabilising the complex and causing Pumilio to bind
extra nucleotides 5′ of the PRE. Thus, the specificity is shifted to the 5′ end
of the sequence and adjacent nucleotides and allows Pumilio to bind imperfect
PREs. The increase in gene repression is dependent on both the number of NREs
in the 3’UTR and the concentration of Nos. However, these results come from research
on Drosophila and similar
interactions are yet to be studied in other species.

Protein and
RNA modifications.

PUF RNA binding can also be affected by post-translational
modification, phosphorylation in particular. The effect of phosphorylation
differs with the PUF: for human Pumilio 1 it increase the proteins affinity for
its’ cognate RNA, while for allow the association of already bound mRNAs with
polysomes, promoting their translation. It is noteworthy that the phosphorylation sites found so far mostly reside in regions outside
the PUF-HD. RNA modifications, such as pseudouridilation (?) and
N6-methyladenylation (m6A) have been shown to decrease PUF affinity.
The extent depends on the number and, in case of m6A, the position
of the modified nucleotides. Curiously, with most of the modification variants
it is the rate of dissociation that is affected. As these protein and RNA
modifications can be reversible, they could be incorporated as an additional
step of control over PUF binding when constructing regulatory networks. However, a lot of study is yet to be
carried out before this becomes practically possible.

outside of the PUF-HD

It has been shown that non-PUF-HD regions of PUF proteins
also can also affect their RNA binding activity. For example, the non-canonical
yeast Puf2p, has RNA recognition motifs and a low complexity region in addition
to its’ PUF-HD domain. These outer regions, while not essential for RNA-binding,
appear to increase the PUFs specificity, as a Puf2p mutants lacking them bind a
greater number of targets, fewer of which have a PRE. Thus, studying RNA-binding behaviour of the whole protein
can yield many novel and potentially useful PUF properties.

used to study PUF RNA binding

A few established techniques have been used traditionally to
study PUF RNA binding. In vivo it is
commonly determined by a yeast 3-hybrid assay, where the PUF, fused to a
transcriptional activator, binds the RNA of interest which is attached upstream
of LacZ and sometimes His3 reporter genes. The interaction and its extent are determined
by the ability to grow on a selective medium and from b-galactosidase activity.
UV-crosslinking also allows finding PUF targets in vivo, as bound RNAs are linked to the PUF and purified. In vitro specificity and affinity can be
inferred using a variation of an electrophoretic mobility shift assay (EMSA), which
can detect binding as the PUF-RNA complex travels slower through the gel medium
than unbound RNA. However, more unusual methods, such as fluorescence
resonance energy transfer (FRET), have also been utilised. This technique
is based on the ability of donor and acceptor fluorophore molecules to transfer
and receive non-radiative energy when in close proximity. Alone, the donor
fluoresces when excited by a laser. However, when it is close to an acceptor,
the excitation energy is transferred, causing the acceptor to fluoresce instead.
If the two fluorophores are attached to an RNA sequence at either end of a PRE,
the distance between them changes depending on whether the PUF is bound. Unlike
previously utilised methods which could only provide apparent Kds, a novel variation
of FRET, single molecule FRET (smFRET), not only allows to see the affinity and
specificity of RBPs, but also their binding dynamics.  This technique involves the immobilisation of
single RNA molecules on a slide and, by monitoring the fluorescent profiles of
the donor and acceptor fluorophores, could be used to observe the formation of
individual PUF-HD-RNA complexes. Thus, this method could be utilised to probe a
previously understudied aspect of PUF RNA binding.


While recent study has revealed many factors affecting the
affinity and specificity of PUF RNA binding, very little is known about the
kinetics of the process. The aim of this project will be to use smFRET to study
the dynamics of PUF-HD binding, such as the association and dissociation rates
and the amount of time the proteins stays bound to its cognate RNA. Additionally,
this project will investigate how these might be affected by temperature, ion
and salt concentrations, mutations within the PUF-HD and possibly the presence
of the Nos binding partner. This has not been previously attempted for Pumilio
proteins and could provide novel and valuable information on the binding
process and presence of possible binding intermediates.


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