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Further work resulted into novel insights in mRNA-protein regulatory networks and led to the development of novel technologies like high-throughput sequencing cross-linking and immunoprecipitation (HITS-CLIP) to study the interactions of microRNA and RNA targets in the brain (and other tissues).

Directed analysis of unbiased HITS-CLIP targets can reveal cryptic biology, as in the analysis of developmental HITS-CLIP maps to study NOVA interactions with transcripts of the Reelin pathway and thereby identify a previously unknown NOVA-dependent alternative exon necessary for proper neuronal migration (Yano et al., 2010).

We designed a factorial fertilization and clipping experiment to study the potential competition between plants and soil microorganisms for soil nitrogen in an ecosystem under grazing.

CLIP was also used to study the function of the RNA-binding protein Rrm4, discovering that Rrm4 may transport RNAs from the nucleus to cell poles [11].

In summary, CLIP and HITS-CLIP are very useful techniques to study protein RNA interactions, which can be applied to different biological samples such as bacteria, fungi, yeast, Caenorhabditis elegans, mammalian tissue including brain, and culture cells including human embryonic stem cells and HeLa cells.

HITS-CLIP was first applied to study Nova RNA interactions and uncovered the 3' end RNA processing rules in the brain [8].

Additionally, HITS-CLIP was also applied to study other RBPs, such as Argonaute in mouse brain [12], polypyrimidine tract-binding protein (PTB, also known as hnRNP I) in HeLa cells [13], FOX2 in human embryonic stem cells [14] and so on.

CLIP has been successfully applied to study protein-RNA interactions in bacteria, fungi, yeast, and mammals [ 4– 10].

CentriMo could also be used with RNA-binding protein (RBP) motifs to study paired CLIP-seq (or equivalent) datasets.

To explore the differences between the nuclear and cytoplasmic roles of NOVA, Eom and colleagues separated brain tissue from mice into nuclear and cytoplasmic fractions, and employed a technique called HITS-CLIP (Licatalosi et al., 2008) to study the binding between the NOVA proteins and the RNA in these two fractions.

All spatial data were clipped to the study area boundary.

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