DISCUSSION
In this article, we present hnRNPA1 recruiting oligonucleotides (AROs),
a new tool for mRNA knockdown consisting of a short targeting
oligonucleotide and a hnRNPA1 recruiting loop. Through a series of
RT-qPCR experiments targeting KRT14 and TBK1, we have shown that AROs
expressed from plasmids are able to knock down the expression of two
independent genes in a targeted fashion. We have shown that both the
antisense targeting RNA and the hnRNPA1 recruiting loop must be present
for an efficient knockdown. We have also shown that AROs function in an
hnRNPA1 dependent manner. Finally, we have shown that AROs are
functional in multiple cell lines including human keratinocytes (HaCat)
and squamous cell carcinoma cells (SCC9) and can affect a
loss-of-expression functional phenotype.
Our results suggest that the location of the sequence targeted by an ARO
on the exon of interest does not have a large influence on knockdown
efficiency. We initially hypothesized that the targeting location would
have a bigger impact on ARO function. Specifically, we expected that it
would be necessary to direct AROs to splice junctions or to ASF/SF2
motifs, as preventing binding of SR splice enhancer proteins is a known
mechanism of hnRNPA1 splice site repression (Jean-Philippe et al., 2013;
Kashima et al., 2007). One possible explanation is the cooperative
recruitment of hnRNPA1 molecules along RNA in the 3’ to 5’ direction
(Jean-Philippe et al., 2013). This might mean that an ARO binding at a
downstream exonic sequence is sufficient to suppress SR protein
recruitment at upstream regions. Another possibility is that the exon we
choose was too small to see location specific effects. Larger exons with
more space between splice sites should be tested in the future to help
answer this question.
A major advantage of AROs over most knockdown technologies is the ease
with which they can transition from promotor-based expression (i.e. from
a plasmid or lentivirus construct) to RNA oligonucleotides that can be
directly delivered to cells or organisms. Here we have shown the utility
of AROs as a potential therapeutic by synthesizing single-stranded RNA
AROs and showing that they are able to achieve similar levels of
knockdown as their plasmid counterparts. By using ssRNA AROs to knock
down KRT14 in SCC9, a squamous cell carcinoma line used as a model for
epithelial-mesenchymal transition studies in head and neck cancer, we
demonstrate that ARO-based knockdown resulted in a biologically relevant
phenotype – the decreased ability of carcinoma cells to migrate in a
cell invasion assay.
An unexpected finding in our RNA oligonucleotide experiments was that
while the double ARL ssRNA (ARL-Oligo-ARL) worked to some degree at high
concentrations, the single ARL (ARL-Oligo) achieved significantly better
gene knockdown at lower concentrations. This contrasted with our plasmid
experiments which found that double ARL AROs performed best. A possible
explanation for this may be that the additional size or complexity of
double ARL oligonucleotides may have lessened their ability to make it
to the nucleus. Another possibility is that complementary regions
between the two ARLs caused secondary structure leading to
double-stranded portions of the construct being degraded in the
cytoplasm. This would be less likely to occur in the plasmid construct
as those AROs would be produced in the nucleus and would presumably have
more opportunity to affect pre-mRNA before being exported and degraded.
We hypothesize that AROs knock down gene expression by causing errors in
pre-mRNA splicing followed by nonsense-mediated decay. However, it is
important to point out that there are several plausible alternative
hypotheses. For example, hnRNPA1 has been shown to bind to the 3’ UTR of
some genes, and this can lead to mRNA deadenylation and degradation
(Geissler et al., 2016). This may be why binding along any point of an
exon in pre-RNA seems to be effective. While more work will need to be
done to determine the specific mechanism of action of AROs to better
understand and improve their function, their utility as biological tools
in their current form remains substantial.
AROs represent a novel paradigm for recruiting RNA binding proteins to
pre-mRNAs via synthetic constructs. Here we have shown that hnRNPA1 can
be recruited via a short targeting oligonucleotide to knock down
transcript levels of a target gene. While more work will need to be done
to understand the exact mechanism of how AROs function, the work here
illustrates their potential as a novel, biologically functional way to
knock down target transcripts through the redirection of endogenous
cellular machinery. Because RNA binding proteins are ubiquitous and
abundant across cell types, their recruitment to targeted pre-mRNAs may
be a useful way to modulate RNA production, modification, or
degradation. The diverse array of functions they perform and their
directable nature make them an ideal source to mine for molecular
biology tools. The ability to express AROs, and similarly designed
molecules, from a Pol II promoter also has substantial advantages. AROs
could be placed under the control of cell or tissue specific promotors
and used in tissue specific studies or placed behind activatable
promotors.
AROs can both be expressed by constructs or used directly as ssRNA
molecules with little modification. This dual functionality puts them in
a unique position to bridge the gap from high-throughput screens to
independently functional molecules. We believe AROs fill an important
niche in the scientific toolbox and we hope the work here can serve as a
template for future constructs that make use of the potential of RNA
binding proteins as tools for the modulation of RNA homeostasis.