3.4 Regulation of miRNA availability miRNA genes are transcriptionally regulated just like any other gene, so their levels in the cell can be controlled through these routes. However, there is one additional way in which the availability of miRNAs within a cell can be regulated. This is through the hybridisation to competitive endogenous RNAs. These RNA chains appear to act as 'sponges', competing with target mRNAs within a cell for binding, and therefore acting as regulators for the effects of a miRNA (Figure 3.6). Figure 3.6 Endogenous RNAs, including circular RNAs, can serve as regulators of miRNAs through competitive binding. Competitive regulation of miRNAs in the cellular pool serves to regulate both the translation blocking effects of 3 UTR binding and the targeting of mRNAs for degradation. Schematic diagram to show how endogenous RNAs can regulate miRNAs through competitive binding. The blue background indicates the cytosol, mRNA are orange strands and the miRNAs are magenta. At the centre is a pool of 6 miRNAs shown, which can target mRNA for degradation (bottom) or bind the 3'UTR of mRNA to block translation of the mRNA (top right). However, this miRNA regulatory role is itself subject to regulation through competitive binding with endogenous RNAs such as the circular RNA pictured (top left). The miRNAs bound to circular RNA are not available to regulate the mRNA until they are released. One of the first well-characterised examples of this was provided by Hansen et al. (2013). These researchers identified a circular RNA chain called ciRS-7 in the mouse brain (see Figure 3.7a, left). The ciRS-7 RNA contains over 70 sites with sequence complementarity to miR 7 (Figure 3.7b). (b) View larger image Figure 3.7 The circular RNA ciRS-7 acts as a sponge, regulating the availability of miRNA miR-7. (a) Both are expressed in the same cells in the brain, as shown by similar patterns of brown staining in mouse brain sections. (b) ciRS-7 carries multiple target sites for miR-7, drawn here to represent the base conservation along ciRS-7 for each site complementary to miR 7 . for miR-7) acting as a sponge to regulate the availability of miRNA miR-7. a. Mouse brain sections stained with a probe to detect ciRS-7 (left) and miR-7 (right). Note the similar distribution of brown staining in both images indicating similar distribution patterns of both miRNA and ciRS-7 in the mouse brain sections (eg the ' C '-shaped brown staining in the hippocampus region; you can ignore the staining intensity differences between the two images in the cortex). b. The orange bar represents the gene for ciRS- 7 gene. Above it is a graph showing degree of similarity to the miR-7 RNA sequence. The taller the hight of the black block, the closer the match. Note that there are over 70 such conserved sites showing sequence complementarity to miR 7 . On the right hand site a dotted box shows the region enlarged (below the orange ciRS-7 RNA bar) which shows many black boxes co.

1. 3.4 Regulation of miRNA availability miRNA genes are transcriptionally regulated just like any
other gene, so their levels in the cell can be controlled through these routes. However, there is
one additional way in which the availability of miRNAs within a cell can be regulated. This is
through the hybridisation to competitive endogenous RNAs. These RNA chains appear to act as
'sponges', competing with target mRNAs within a cell for binding, and therefore acting as
regulators for the effects of a miRNA (Figure 3.6). Figure 3.6 Endogenous RNAs, including
circular RNAs, can serve as regulators of miRNAs through competitive binding. Competitive
regulation of miRNAs in the cellular pool serves to regulate both the translation blocking effects
of 3 UTR binding and the targeting of mRNAs for degradation. Schematic diagram to show how
endogenous RNAs can regulate miRNAs through competitive binding. The blue background
indicates the cytosol, mRNA are orange strands and the miRNAs are magenta. At the centre is a
pool of 6 miRNAs shown, which can target mRNA for degradation (bottom) or bind the 3'UTR
of mRNA to block translation of the mRNA (top right). However, this miRNA regulatory role is
itself subject to regulation through competitive binding with endogenous RNAs such as the
circular RNA pictured (top left). The miRNAs bound to circular RNA are not available to
regulate the mRNA until they are released. One of the first well-characterised examples of this
was provided by Hansen et al. (2013). These researchers identified a circular RNA chain called
ciRS-7 in the mouse brain (see Figure 3.7a, left). The ciRS-7 RNA contains over 70 sites with
sequence complementarity to miR 7 (Figure 3.7b). (b) View larger image Figure 3.7 The circular
RNA ciRS-7 acts as a sponge, regulating the availability of miRNA miR-7. (a) Both are
expressed in the same cells in the brain, as shown by similar patterns of brown staining in mouse
brain sections. (b) ciRS-7 carries multiple target sites for miR-7, drawn here to represent the base
conservation along ciRS-7 for each site complementary to miR 7 . for miR-7) acting as a sponge
to regulate the availability of miRNA miR-7. a. Mouse brain sections stained with a probe to
detect ciRS-7 (left) and miR-7 (right). Note the similar distribution of brown staining in both
images indicating similar distribution patterns of both miRNA and ciRS-7 in the mouse brain
sections (eg the ' C '-shaped brown staining in the hippocampus region; you can ignore the
staining intensity differences between the two images in the cortex). b. The orange bar represents
the gene for ciRS- 7 gene. Above it is a graph showing degree of similarity to the miR-7 RNA
sequence. The taller the hight of the black block, the closer the match. Note that there are over 70
such conserved sites showing sequence complementarity to miR 7 . On the right hand site a
dotted box shows the region enlarged (below the orange ciRS-7 RNA bar) which shows many
black boxes complementary to miR-7. Note the solitary white box denotes similarity to another
miR (miR-671) which when bound to ciRS-7 causes the circular RNA to be degraded. (From
Hansen et al. (2013)). In examining the mouse brain, it is clear that miR- 7 is co-expressed in a
subset of the cells that contain ciRS-7, such as the cells of the mouse hippocampus (see Figure
3.7a, right: the 'C'-shaped group of brown-stained cells that stain for ciRS-7 in Figure 3.7a, left).
Thus, both the miR and sponge are expressed in the same cells. When bound to miR-7, the ciRS-
7 RNA is not degraded until it is bound by another miRNA called miR-671, for which one
binding site exists. Binding of miR-671 directs degradation of the circular RNA and any bound
miRNAs in an argonaute-dependent manner. Thus, in this case, one RNA chain that regulates the
availability of miRNAs is, itself, regulated by a miRNA. The example of circular RNAs acting as
molecular sponges and sequestering miRNAs provides another example of the interdependence
of RNA molecules in the cell. The ability to base-pair with multiple complementary targets
allows for a highly integrated and dynamic network to develop. Changes in the levels of: -
miRNAs - a competitive endogenous regulator ('sponge') - other mRNAs with matching target

2. sites all serve to regulate the availability of miRNAs available to direct translational repression
or degradation. This level of cross-talk is not achievable with proteins and illustrates the
plasticity of RNA as a regulator molecule. Until now, the focus has been on events occurring
within a single cell. One additional aspect of miRNAs that will be considered next is their use as
transcellular regulatory molecules. Changes in the levels of: - miRNAs - a competitive
endogenous regulator ('sponge') - other mRNAs with matching target sites all serve to regulate
the availability of miRNAs available to direct translational repression or degradation. This level
of cross-talk is not achievable with proteins and illustrates the plasticity of RNA as a regulator
molecule. Until now, the focus has been on events occurring within a single cell. One additional
aspect of miRNAs that will be considered next is their use as transcellular regulatory molecules.
This question carries 10% of the marks for this assignment and assesses module learning
outcomes KU1, KU4 and CS1-2. This question relates to Topic 5 (Part 3). a. Briefly describe the
ways in which the availability of a mature miRNA can be regulated in a eukaryotic cell. (5
marks) b. The coding strand of the genomic DNA base sequence of a region that is transcribed to
form the plant miR171b primary RNA is shown graphically in Figure 1. The bases that encode a
mature 21 nucleotide miR are shown in blue and the corresponding region to which it pairs to
form the pre-miR (pre-miRNA) is shown in red. Figure 1 Region of genomic DNA that is
transcribed to form the plant miR171b primary RNA. Draw a schematic figure of the secondary
structure for miR primary RNA transcribed from this gene. You should show the base sequence
of the miR (blue) and the sequence to which it base-pairs (red), and indicate which bases are
base-paired in the double-stranded paired region.