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The three information-carrying biomolecules, DNA, RNA and protein, interact extensively to coordinate their behaviors in the cells. The dynamic assembly of DNA-RNA-protein macro- molecular complexes is critical for executing hardwired genetic programs and responding to extracellular stimuli. Therefore, comprehensive knowledge of the diversity of molecules and their intricate interactions is the foundation for modern molecular biology.

Technological innovation is a driving force for scientific discoveries. Numerous biochemical techniques have been developed in the past decades to study the interactions among DNA, RNA and proteins. With these methods, we are beginning to have a rough overview of how macromolecular machineries are organized in space and time. The methods generally have the same principles and have gradually evolved from low throughput to high throughput. Here I briefly review the biochemical methods developed so far for studying interactions, with a focus on the high throughput varieties. The commonly used methods are summarized in Table 1.1. Genetic methods for high throughput detection of interactions, like yeast two-hybrid screens, are not discussed here.

Traditional ways to analyze DNA-RNA-protein interactions usually involve the purification of individual components using a variety of chromatography or immunological methods. These methods take advantage of the properties of biomolecules, for example, sequence complemen- tarity in DNA and RNA sequences, antibody-antigen affinities etc. Once one or both molecules we try to assay are purified (not necessarily to homogeneity), several types of methods are used to detect their interactions, for example, electrophoretic mobility shift assay (EMSA), co-immunoprecipitation (co-IP), pull-downs, etc.

EMSA and related methods can be used to detect the interactions between protein and protein, DNA and protein, RNA and protein. Co-IP and related methods can also be used for these purposes. Subsequent detection of molecules could involve a variety of methods, like non- specific dye staining (using Coomasie Blue etc.), western blotting, northern blotting, southern blotting, PCR, etc.

Known Unknown Low  throughput High  throughput Note

DNA DNA Conforma+on  capture  w/  PCR 3C,  4C,  5C,  hi-­‐C  via  chip/seq

DNA RNA enChIP-­‐MS

DNA Protein EMSA,  etc. enChIP-­‐MS

RNA DNA ChIRP-­‐PCR,  etc. ChIRP-­‐seq

RNA RNA EMSA  like,   CRAC,  CLIP  (for  Ago),  and  to  be  developed RNA Protein EMSA,  RNA  pulldown.   Interactome  capture,  ChIRP-­‐MS

Protein DNA EMSA,  IP-­‐PCR ChIP-­‐chip/seq,  ChIP-­‐exo,  na+ve-­‐ChIP,  etc. Protein RNA EMSA,  IP-­‐PCR RIP-­‐chip/seq,  CLIP  variants

Protein Protein co-­‐IP,  pulldown,  etc. protein  array,  IP-­‐mass  spec,  etc.

Table 1.1: Biochemical methodologies for studying DNA-RNA-Protein interactions.

This is not a comprehensive list, as less commonly used methods are not listed here. 3C: chro- mosome conformation capture (Dekker et al., 2002). 4C: 3C on a chip, or circular 3C (Zhao

et al., 2006). 5C: carbon-copy 3C (Dostie and Dekker, 2007). Hi-C: (Beltonet al., 2012).EMSA: electrophoretic mobility shift assay. IP: immunoprecipitation. ChIP: chromatin immunopre- cipitation (Gilmour and Lis, 1984). enChIP: engineered ChIP (Fujitaet al., 2013). ChIP-exo: ChIP with exonuclease treatment (Rhee and Pugh, 2011). RIP: RNA immunoprecipitation (Keene et al., 2006; Zhao et al., 2010; Lu et al., 2014). CLIP: cross-linking immunoprecipita- tion (Ule et al., 2003). PAR-CLIP: Photoactivatable-Ribonucleoside-Enhanced CLIP (Hafner

et al., 2010). MS and Mass spec: mass spectrometry. ChIRP: chromatin isolation by RNA purification (Chu et al., 2012). CHART-seq capture hybridization analysis of RNA targets by sequencing (Simon et al., 2011). CRAC: cross-linking and analysis of cDNAs (Granneman

et al., 2009). ATAC-seq: assay for transposase-accessible chromatin using sequencing (Buen- rostroet al., 2013).

The major disadvantage of these low throughput methods is that, besides the often time- consuming and labor-intensive procedure, they cannot be used to easily discover new types of interactions. The advent of high throughput methods solved this problem. While the molec- ular complex purification step remains similar, the methods for detection of potential binding partners have changed. Mass spectrometry has been developed to identify the proteins that in- teract with specific protein, RNA or DNA in a unbiased way. Microarrays and high throughput sequencing methods have been developed to identify DNA and RNA sequences that interact with other DNA, RNA and proteins. While many possible methods have been developed, how- ever, other approaches remain to be developed, or further optimized; for example, a method for identifying all intermolecular RNA interactions, a method for identifying all protein and RNA components associated with a specific RNA. The development of these methods, while

challenging, would greatly advance studies on RNA-RNA and RNA-protein interactions. Fur- ther ideas on these directions and potential applications will be discussed in the Conclusions chapter.

Critical to the interpretation of these analyses are the inclusion of proper controls and inde- pendent lines of evidence. However, these principles, which are widely applied to research, have been largely neglected in the execution and analysis of many high throughput experiments. Many of the genome-wide studies tend to report a large number of identified molecules, with little confidence. In other words, people have inappropriately placed more emphasis on sensitiv- ity than on specificity (see a nice layman’s summary of things in genomics approaches that need special attention here, http://genomeinformatician.blogspot.co.uk/2011/07/10-rules-of-thumb- in-genomics.html). A recent paper actually showed that almost none of the PAR-CLIP datasets have proper controls (Friedersdorf and Keene, 2014). With this in mind, we included proper controls and multiple lines of experiments in our RIP-seq analysis of Sm proteins (in Chapter 2) (Luet al., 2014).