specific miRNA mimic or the stable introduction of a miRNA expression construct by a
lentiviral vector. Conversely, miRNA inhibition can be achieved by chemically modified RNA analogues, such as anti-miRs [84], antagomiRs [85] and miRNA sponges
[86], able to bind and block mature miRNAs. Reporter assay is used to validate
individual miRNA:mRNA interactions, measuring the activity or expression of
reporter protein [87]. Various computational and pull-down methods have been developed to improve the identification of the direct association of miRNA-target hybrids,
including RNA immunoprecipitation of ectopically expressed components of the
RNA-induced silencing complex or the affinity purification of synthetic miRNAs of
interest, although the physiological significance of most identified miRNA-target
associations is not still well understood [88]. Interestingly, newly developed techniques
such as CLIP [89] and photoactivatable ribonucleoside-enhanced cross-linking and
immunoprecipitation (PAR-CLIP) [90, 91] investigate the indirect relationships
between miRNAs and their targets, combining the stabilization of protein-RNA complexes by ultraviolet (UV)-induced cross-linking with HTS (Fig. 9). Specifically, the
CLIP method is able to define interaction sites by isolating and sequencing of small
RNA segments cross-linked to RNA binding proteins (RBPs) [92]. However, this
approach has some limitations, such as low RNA-protein cross-linking efficiency by
exposure to 254 nm UV light and high background noise, requiring several control
cross-linking experiments to correctly detect the isolated RNA fragment. Some of these
problems were addressed by PAR-CLIP [90]. The protocol includes the incubation of
cells with medium enriched with a photoactivatable nucleoside, such as 4-thiouridine
and 6-thioguanosine, improving UV cross-linking [93, 94]. Irradiation of the cells by
UV light of 365 nm, which is less harmful than 254 nm for in vivo experiments, triggers
the cross-linking of photoreactive nucleoside-labelled cellular RNAs to interacting
RBPs. Thus, PAR-CLIP is able to enhance RNA recovery and specificity of crosslinking, achieving single-nucleotide resolution of the binding site [95]. These
approaches provide a large amount of molecular information on miRNA targets
identified in RBP complexes, but the detailed protocol is time-consuming and costly
and requires very specialized data analysis tools. In addition, the identification of
miRNAs regulating specific mRNAs remains limited due to the imperfect complementarity of miRNA:mRNA transcripts. Interestingly, miRNA Trapping by RNA in vitro
affinity purification (miR-TRAP) attempts to overcome this limitation, providing an
evolved in vitro RNA affinity purification protocol for the rapid capture of the complex
containing the trapped miRNA/target mRNA pair in the specific cellular context of
choice [96]. The advanced miR-TRAP protocol includes photoactivatable ribonucleosides on transfected miRNA to allow only for complexes containing specific miRNAs
and higher wavelength cross-linking, which is preferable for in vivo studies [96]. Specifically, the miRNA is conjugated to psoralen to produce a highly photoreactive probe.
When the cells are exposed to UVA light, the Pso moiety of the miRNA reacts with
uridine on target mRNAs. Pso-modified miRNA mimics act similarly to endogenous
miRNAs, eliminating the use of antibodies, which can create non-specific background
signals and complicate data interpretation. The cross-linking between the tagged
miRNAs and target mRNAs is then stringently purified by streptavidin-coated beads,
552
C. Dell’Aversana et al.
lentiviral vector. Conversely, miRNA inhibition can be achieved by chemically modified RNA analogues, such as anti-miRs [84], antagomiRs [85] and miRNA sponges
[86], able to bind and block mature miRNAs. Reporter assay is used to validate
individual miRNA:mRNA interactions, measuring the activity or expression of
reporter protein [87]. Various computational and pull-down methods have been developed to improve the identification of the direct association of miRNA-target hybrids,
including RNA immunoprecipitation of ectopically expressed components of the
RNA-induced silencing complex or the affinity purification of synthetic miRNAs of
interest, although the physiological significance of most identified miRNA-target
associations is not still well understood [88]. Interestingly, newly developed techniques
such as CLIP [89] and photoactivatable ribonucleoside-enhanced cross-linking and
immunoprecipitation (PAR-CLIP) [90, 91] investigate the indirect relationships
between miRNAs and their targets, combining the stabilization of protein-RNA complexes by ultraviolet (UV)-induced cross-linking with HTS (Fig. 9). Specifically, the
CLIP method is able to define interaction sites by isolating and sequencing of small
RNA segments cross-linked to RNA binding proteins (RBPs) [92]. However, this
approach has some limitations, such as low RNA-protein cross-linking efficiency by
exposure to 254 nm UV light and high background noise, requiring several control
cross-linking experiments to correctly detect the isolated RNA fragment. Some of these
problems were addressed by PAR-CLIP [90]. The protocol includes the incubation of
cells with medium enriched with a photoactivatable nucleoside, such as 4-thiouridine
and 6-thioguanosine, improving UV cross-linking [93, 94]. Irradiation of the cells by
UV light of 365 nm, which is less harmful than 254 nm for in vivo experiments, triggers
the cross-linking of photoreactive nucleoside-labelled cellular RNAs to interacting
RBPs. Thus, PAR-CLIP is able to enhance RNA recovery and specificity of crosslinking, achieving single-nucleotide resolution of the binding site [95]. These
approaches provide a large amount of molecular information on miRNA targets
identified in RBP complexes, but the detailed protocol is time-consuming and costly
and requires very specialized data analysis tools. In addition, the identification of
miRNAs regulating specific mRNAs remains limited due to the imperfect complementarity of miRNA:mRNA transcripts. Interestingly, miRNA Trapping by RNA in vitro
affinity purification (miR-TRAP) attempts to overcome this limitation, providing an
evolved in vitro RNA affinity purification protocol for the rapid capture of the complex
containing the trapped miRNA/target mRNA pair in the specific cellular context of
choice [96]. The advanced miR-TRAP protocol includes photoactivatable ribonucleosides on transfected miRNA to allow only for complexes containing specific miRNAs
and higher wavelength cross-linking, which is preferable for in vivo studies [96]. Specifically, the miRNA is conjugated to psoralen to produce a highly photoreactive probe.
When the cells are exposed to UVA light, the Pso moiety of the miRNA reacts with
uridine on target mRNAs. Pso-modified miRNA mimics act similarly to endogenous
miRNAs, eliminating the use of antibodies, which can create non-specific background
signals and complicate data interpretation. The cross-linking between the tagged
miRNAs and target mRNAs is then stringently purified by streptavidin-coated beads,
552
C. Dell’Aversana et al.