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Page 6 of 10 Grelet et al. J Cancer Metastasis Treat 2019;5:16 I http://dx.doi.org/10.20517/2394-4722.2018.85

Figure 2. Molecular mechanism of hnRNP E1-mediated translational silencing. The eukaryotic elongation factor-1A1 (eEF1A1) forms
a complex with hnRNP E1 and the BAT element, and silences specific protein expression by stalling the elongation of their translation
by ribosomes. Given the necessity for cognate-codon interaction with the ribosomal A site, it is likely that the formation of the BAT
mRNP complex occurs post-delivery of the aminoacyl-tRNA to the ribosome. The ability of the BAT mRNP complex to inhibit eEF1A1-
dependent elongation suggests that the 3’-UTR is interacting with the 5’-UTR in a circularized model to facilitate its proximity to the 80S
ribosome [35] . It has been suggested that translatable mRNAs are likely to be found in circular forms due to interaction between PABP,
eIF4G, and the cap-binding protein eIF4E [56]

Indirect control of translation through alternative splicing
[57]
Alternative splicing regulates over 90% of multi-exon protein-coding genes in humans . Abnormal
regulation of alternative splicing often produces disease-specific protein isoforms [58,59] . Additionally, genome-
wide analysis has identified tens of thousands of “splice variant” mRNAs that are enriched in a wide range
of human diseases [60,61] . The hnRNP E1 protein is well documented for its repressive role in alternative
splicing mechanisms as they apply to human health and disease. For instance, hnRNP E1 represses tumor
[62]
cell invasion by inhibiting the alternative splicing of CD44 . It was therefore demonstrated that enforced
hnRNP E1 expression inhibited CD44 variants expression in HepG2 human liver cancer cells while
[62]
knockdown of endogenous hnRNP E1 induced these variants splicing . Interestingly, another study based
on in vitro and in vivo models of breast cancer progression demonstrated the switch of CD44 expression
occurring from variant isoforms to the standard isoform during EMT. This isoform switch to CD44s was
essential for cells to undergo EMT and was required for the formation of breast tumors that display EMT
[63]
characteristics in mice . HnRNP E1 also binds to the growth hormone receptor pseudoexon and prevents
[64]
its usage to allow the expression of a functional protein . Disruption of hnRNP E1 binding and subsequent
activation of an alternative splicing event responsible for Laron syndrome was demonstrated either by
hnRNP E1 knockdown or by alterations to the genomic pseudosite. We also recently reported the binding
of hnRNP E1 to a pre-RNA pseudosite in the serine/threonine-protein phosphatase 1 regulatory subunit 10
[30]
(PNUTS) transcript . The hnRNP E1 protein binds to a conserved BAT element that is similar in structure
to those observed in the 3’-UTRs of the mesenchymal encoding mRNAs discussed above. The loss of hnRNP
E1 binding to the alternative splicing site of PNUTS following hnRNP E1 knockdown, phosphorylation,
and/or cytoplasmic translocation activates usage of the pseudosite and generates an alternatively spliced
isoform of PNUTS. This alternative PNUTS isoform does not encode a functional protein, but rather a non-
coding isoform of the gene. Functionally, the lncRNA-PNUTS acts as a decoy for miRNA-205 and binds
to the miRNA causing a decrease in miRNA-205 bioavailability. This abolishes translational inhibition
[30]
of mesenchymal factors such as ZEB mRNAs that would otherwise be suppressed in epithelial cells .
Since alternative splicing and generation of lncRNA-PNUTS is an early event in TGFß-mediated EMT,
the lncRNA-PNUTS likely operates as a transient inhibitor of the miRNA-205 to allow for the temporal
upregulation of ZEBs and subsequent regulation of downstream EMT events. Indeed ZEBs proteins are
reciprocally linked in a feedback loop with the miR-200 family, each strictly controlling the expression of the
other [18,65] . In this way, a transient, but nevertheless, strong decrease in miR-205 bioavailability, sufficient to
activate the ZEB proteins, would allow for transcriptional repression of the miR-200 family or other miRNAs