Discussion
The importance and functionality of RNP motifs in RBP-RNA interactions
are well established in previous studies [9]. Since there is a high
sequence similarity between NCL and hnRNP A1 RBDs, and hnRNP A1-RNA
interactions are known, a preliminary prediction of putative residues
that may be important in NCL-miRNA interactions could be made based on
sequence conservation between NCL and hnRNP A1. The RBP-hnRNP A1
interacts with MPC and pri-mir-18 by utilizing aromatic/charged residues
found on its RNP motifs as well as on non-RNP beta strands [9].
These aromatic/charged residues show strong conservation between NCL and
hnRNP A1 sequences suggesting existence of a similar RNP-mediated
mechanism in NCL-RNA interactions (Fig 6A ). This study confirms
this prediction through detailed docking analyses in silico . Our
results provide three alternative NCL-miRNA binding possibilities with
common underlying conserved residues at an equivalent position as in
hnRNP A1-RBD taking part in RNA binding (Fig 6C ). Aromatic
residues in the RNP motifs are known to be capable of initiating
interactions with multiple nucleobases at the same time through stacking
interactions and promoting structural stability to the RNA-RNP binding
motif [10]. We consistently predicted the involvement of these
aromatic residues in all binding modes of miRNA-NCL. Additionally, we
frequently predicted some charged residues on different beta strands
from the same RBDs to be involved in NCL-miRNA interactions. Several
arginine residues (R291, R293, and R298) were consistently predicted to
interact with phosphate groups on the nucleotides via salt bridges
(e.g., R293 with G16 in Fig 3, and with A34, and U45 in the figures for
mir-16, mir-221, and mir-222, respectively). These residues provide
additional structural stability to RNA-protein interactions similar to
previous studies [9]. Our docking results also revealed that each
NCL-RBD interacts with the miRNA duplex structure from opposite sides
and forms a clasp around it. Linker regions between RBD3 and RBD4 were
consistently predicted in several scenarios to hold the miRNA molecule
from an additional third side, thus tightening the NCL-RBDs grip on
miRNA (Fig 4-6 ).
Our results indicate that NCL RBDs demonstrate preference or affinity
for interaction with certain types of miRNA motifs. It was previously
established that NCL prefers interacting with RNA loops while driving
ribosomal biogenesis [42]. Non-canonical base pairing is known to
lead to kink-turns, bulges, mismatched pairs, and wobble pairs in miRNA
structures and presents suitable interaction sites that would be
unavailable otherwise. In canonical pairs such as G-C or A-U, amino
groups of each base-pair are projected into the major groove, creating a
region with positive electrostatic potential [43]. The G-U base-pair
is an example of non-canonical base pair, where oxygen groups from both
nucleotides face the major groove side instead of the amino groups and
leading to negative electrostatic potential in the region of the major
groove of the dsRNA [43]. Our results also highlighted the
preference of NCL RBDs on non-canonical base pairs when interacting with
miRNAs (Supporting Figure S5 ). Such regions are expected to
interact with amino acids with positively charged side groups such as
arginine or lysine [43]. This ties in with our results as several
arginine and lysine residues from NCL RBD3-4 were predicted as some of
the most frequently encountered residues in the various docking
scenarios obtained.
Additionally, wobble pairs and mismatched pairs are known to be
important elements of primary miRNA processing by the MPC [44].
Certain RNA motifs such as UGU/GUG are known to be enriched around the
apical loop regions [45] and the preference of NCL to interact with
regions close to the apical loops has been demonstrated in previous
studies investigating NCL-rRNA [42] and NCL-mRNA [26]
interactions. This consensus sequence is also important in pri-miRNA
processing by the MPC as DGCR8 is thought to recognize and interact with
this consensus sequence [46]. Our results revealed that NCL RBDs
were able to recognize this UGU/GUG motif for all miRNA molecules tested
in this study (Fig 7 ). In the cases of mir-15a and mir-103a,
these regions were identified adjacent to apical loop structures. It is
also interesting to note that, in binding mode 2 (observed in mir15a and
mir103a), RBD4 by itself seems sufficient to drive NCL-miRNA
interactions (Fig 7A ). In the cases of mir-21 and mir-16-1, NCL
interacts with regions distant from apical loops, but closer to bulge
regions towards the middle of the miRNA molecule. (Fig
7B ). Since DGCR8 also recognizes the same motif for
interactions, our results suggest that NCL potentially binds adjacent to
DGCR8 or could even replace it in as a possible scenario of miRNA – NCL
interactions. Alternatively, in the cases of mir-221 and mir-222,
NCL-miRNA interactions were predicted to be localized slightly distant
to the apical loop region and closer to the basal stem region containing
a mismatched GHG/CUC motif (Fig 7C ). This manifests as a
mismatched bulge, which is a common element in most pri-miRNA structures
[46]. Drosha cleaves its miRNA targets around the basal stem close
to this motif [46]. This cooperation model suggests that NCL RBDs
binds closer to Drosha. In both scenarios, these motifs likely serve as
anchoring points for NCL RBDs when they interact with miRNA in
cooperation with MPC.
Similarly, multiple other RBPs including hnRNP A1, Lin28B, RBFOX3, and
HuR interact with either the terminal loop or the stem regions of the
pri-miRNA molecules to either promote or suppress pri-miRNA processing
by the MPC proteins depending on the location of the interactions and
the targeted miRNA [47]. For example, both Lin28B and hnRNP A1
interact with the terminal loop of a subset of pri-miRNA structures.
hnRNP A1 is predicted to help with the processing of pri-miR18-a by
binding to the terminal loop and causing a relaxation of the miRNA
structure and therefore making it easier for MPC to interact with miRNA
[48]. However, when hnRNP A1 interacts with let-7 pri-miRNA terminal
loop, it outcompetes binding of another RBP, KH-type slicing regulatory
protein (KSRP) known to promote biogenesis of let-7 [48] and
decreases pri-miRNA processing by Drosha. Lin28B, on the other hand,
always negatively regulates this process by interacting with the
terminal loop and inhibiting Drosha from interacting with miRNA
[49]. Both RBFOX3 and HuR bind to the basal stem and inhibit miRNA
processing by blocking catalytic activity of Drosha [50,51]. Based
on these studies, it is clear that the relationship of RBPs with the MPC
is both location and context dependent.
A recent structural study investigating interactions of the MPC with
pri-miR16-2 revealed that 2 DGCR8 proteins interact with nucleotides
adjacent to the apical loop region of the pri-miRNA molecule [52].
Canonical function of DGCR8 is to interact with pri-miRNA using its
double stranded RNA binding domains (dsRBDs) and present the pri-miRNA
molecules to Drosha for processing [52]. This is illustrated inFig 8A. Many RBPs can interact with regions of pri-miRNA
molecules that are also regions where DGCR8 proteins bind. We speculate
that NCL-RBDs promotes pri-miRNA processing of certain miRNAs by a
similar mechanism as observed in hnRNP A1-mir18 interactions. As a RBP
capable of binding double stranded RNA molecules, NCL could potentially
replace the pri-miRNA presenting functions of one or both DGCR8
proteins. Our results suggest that for certain miRNA, NCL can wrap
around the double stranded miRNA molecule with two RBDs like DGCR8
(Fig 8B ). Since the lengths of oncogenic miRNA transcripts are
variable, we speculate that NCL may act as a bridging agent between
DGCR8 and Drosha when processing longer transcripts (Fig 8C ).
Our findings present a snapshot of NCL-miRNA that give an initial
insight into these interactions. We envision future studies to elaborate
the NCL-miRNA interaction dynamics over longer time-scales to get a
finer grained picture of the underlying molecular mechanisms and their
downstream effects.
Drosha and DGCR8 interact with different RNA types including
precursor-mRNA (pre-mRNA) and non-coding RNA and are also involved in
double stranded DNA break repair mechanisms [53]. Since NCL and MPC
proteins can interact in a non-miRNA context [21], it is quite
likely that they cooperate in other biological pathways as well. Studies
testing NCL- MPC cooperation in pre-mRNA processing may be a natural
extension of this study since NCL is also known to interact with mRNA
UTR molecules to manipulate their expression levels in human cancers
[20,25,26].
NCL cellular localization is highly complex and context-dependent.
Pathophysiological functional implications of NCL are even more
enigmatic. Despite this multidimensional behavior, NCL is a promising
target for cancer therapeutics. Therapeutics such as the immuno-agents
4LB5-HP-RNase [54], G-rich DNA oligonucleotide, aptamer AS1411
[55], and antagonist pseudopeptides, N6L [56] and HB19 [57],
target either surface or cytoplasmic NCL to regulate its functions in
miRNA synthesis, RNA metabolism, cell proliferation, angiogenesis and
metastasis, in a variety of cancer types including breast cancer.
In summary, we predict two putative binding modes where NCL RBD3-4
specifically drive NCL-miRNA interactions and an alternative binding
mode where a small contribution from RBD12 is also involved. As we had
hypothesized RNP motifs on RBD3-4 play a significant role in these
interactions; additionally, we report novel residues important for the
interaction that have not been identified in any other previous study.
Importantly, our data support an idea that the exclusive presence of NCL
RBD3-4 in animals might be evolutionarily relevant for NCL functions as
drivers for microRNA processing. Our data delineate critical residues in
NCL and recognition motifs in the miRNA important for NCL-miRNA
interactions. Future studies designed to validate critical residues in
these motifs that are important for interactions, using site-directed
mutagenesis in cellular conditions will be required. Once confirmed
experimentally, NCL-miRNA interacting interface provide a valuable drug
targets for the development of cancer therapies to control specific gene
expression.