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Non-coding RNAs play a big part in epigenetics regulation of gene expression in addition to their roles at the transcriptional and post-transcriptional level.
Recent studies have revealed that about 90% of the eukaryotic genome is transcribed. Interestingly, only 1–2% of these transcripts encode for proteins; the majority are transcribed as ncRNAs.
Non-coding RNAs can be divided into two main types: infrastructural and regulatory ncRNAs. Infrastructural ncRNAs seem to have a housekeeping role in translation and splicing and include species such as ribosomal, transfer and small nuclear RNAs. Regulatory ncRNAs are more interesting from an epigenetic point of view as they are involved in the modification of other RNAs. They can be further classified into the following:
miRNAs are small single-stranded molecules (20–24 nt) that derive from transcripts forming distinctive hairpin structures called pre-miRNA. The hairpin is processed into mature miRNA and forms the RNA induced silencing complex (RISC), which contains miRNA-interacting proteins such as Dicer. The miRNAs will pair with complementary sequences on target mRNAs transcripts through the 3’UTR, leading to gene silencing of the target.
Piwi-interacting RNAs (piRNAs)
piRNAs are small ncRNA (24–31 nt) that are able to form complexes with Piwi proteins of the Argonaute family. piRNAs are characterized by a uridine at the 5’end and a 2’-O-methyl modification at the 3’ end. Their main role is the silencing of transposable elements during germ line development.
Small interfering RNAs (siRNAs)
siRNAs are long linear dsRNA processed by Dicer into mature 20–24 nt siRNAs that direct silencing when loaded onto RISC. They mediate post-transcriptional silencing similarly to miRNA by a process called RNA interference (RNAi), where siRNA interferes with the expression of a complementary nucleotide sequence.
Long non-coding RNAs (lncRNAs)
lncRNAs are considered as non-protein coding transcripts >200 nt in length. The majority of non-coding RNAs belong to this group. Many of the lncRNAs are subject to splicing, polyadenylation and other post-transcriptional modifications, and can be classified according to their proximity to protein coding genes. A subgroup of lncRNAs, named large intergenic non-coding RNAs (lincRNAs) are marked by trimethylation of K4 on histone H3 (H3K4me3) at their promoter and trimethylation of K36 on histone H3 (H3K36me3) along the transcribed region. LincRNAs are involved in epigenetic gene silencing, such as the role of Xist (X-inactive specific transcript) and in tumor development by promoting expression of genes involved in metastasis and angiogenesis.
Enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs)
These two types of ncRNAs have been described recently and their roles are still unclear. Enhancer RNAs are non-coding transcripts with an average of 800 nt (ranging between 0.1–9 kB) and they are produced from regions enriched with monomethylated lysine 4 on H3 (H3meK4), RNA Pol II and coactivators such as p300, which differentiates them from lncRNAs. It has been postulated that eRNAs function as transcriptional activators. PARs are non-coding transcripts that range from 16 nt to 200 nt, and they are generally expressed near the transcription start site or in upstream elements of the promoter. Most of the PARs are associated with highly expressed genes, but they are weakly expressed and with short half-lives. It has been postulated that PARs are connected with transcriptional activation and repression.
Interest in RNA-protein interactions is increasing as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and epigenetic regulation of gene expression by ncRNAs.
The two most common techniques to study RNA-protein interactions are called RIP (RNA Immunoprecipitation) and CLIP (UV Cross-Linking and Immunoprecipitation). Both are variations of the more popular protocol to study DNA-protein interactions called chromatin immunoprecipitation (ChIP).
RIP is used to map RNA-protein interactions in vivo by immunoprecipitating the RNA binding protein of interest together with its associated RNA.
The purified RNA-protein complexes can be then separated by performing an RNA extraction and the identity of the RNA can be determined by RT-PCR. As research into RNA in the last decade has intensified, so has the interest in RIP, leading to development of better RIP protocols.
CLIP is a related technique that differs from RIP in the use of UV radiation to cross-link RNA binding proteins to the RNA that they are bound to. This covalent bond is irreversible, allowing stringent purification conditions to remove non-specific contaminating RNA. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA. CLIP is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins.