Structural Biochemistry/Nucleic Acid/RNA/Small RNA

Small RNA is a classification of RNA which includes small-interferring RNA (siRNA), micro RNA (miRNA), and piwi-interacting RNA (piRNA). These small RNA play important roles in biological and diseases processes.

RNA interference in cultured cells.

siRNA & micro RNA

small-interferring RNA (siRNA) is a class of RNA molecules that are around 20-25 nucleotides in length. They are mostly involved with the RNA interference (RNAi) pathway in order to interfere with the expression of a specific gene.

siRNA is a type of double stranded RNA that was found target mRNA cleavage sites and were designed to target transcript silencing through transfection of the siRNA into mammalian cells. This allowed for the development of RNAi-based applications such as a new class of therapeutics.

micro RNA (miRNA) is a class of RNA molecules that are found in eukaryotic cells. They are generally 20-25 nucleotides in length and are also involved in translation repression and gene silencing. They were similar to siRNA and was found to negatively regulate expression of target transcripts.

The stem-loop of a pre-microRNA.

These two types of RNA were established as guides in governing silencing of target transcripts. This also raised questions of how these small RNAs were produced and it was found that immunoprecipitates in Drosophilia S2 cells processed the dsRNA (double stranded RNA) into the siRNA in vitro. miRNA was found to be derived from a conserved stem-loop precursor. This suggests that a dicing step could be required for miRNA biogenesis. The stem-loop forms part of a several hundred nucleotide long miRNA precursor which is then transcribes into miRNA. The existence of this precursor was found in Drosophia pupae.

In analyzing small RNA pathways in Drosophia, it was found that isolated dicer-1 and dicer-2 mutatnts were responsible for the biogeniss of miRNA and siRNA, respectively. Dicer-1 processed pre-miRNA independent of ATP while Dicer-2 processed dsRNA as ATP dependent. However, in mammalian cells, only one dicer generates both miRNA and siRNA.

siRNA effected silencing as they program RNAi effectors (such as RISC) to target mRNA. RISC is a magnesium dependent endoribonuclease that is affected by miRNA and siRNA to target mRNA cleavage activity.

miRNA has a controversial effector mechanism. This disparity is because there is a lack of a comparable well defined biochemical readout for miRNA induced RISC activity while there is a clear one from siRNA.

Making RISC

RISC: the effector complex for small RNAs

It is known that small RNAs aid in the regulation of gene expression. However, small RNAs cannot function individually to catalyze reactions. Instead, they come together and form RNA-induced silencing complexes (RISCs) in order to help with silencing genes and locating RISC targets. In this sense, the assembly of RISC is crucial for the small RNAs to do their job.

Argonaute: the core component of RISC

The Argonaute (Ago) family of proteins is a main component of RISC that is essential to RISC’s function of target recognition and silencing. The Ago family can be divided into the Ago subfamily and Piwi subfamily. These Ago proteins, each with their own characteristics, are in charge of the functions of the small RNAs that they are paired with. SiRNAs and micro RNAs bind to Ago proteins while piRNAs bind to Piwi proteins. In mammals, the four proteins from the Ago subfamily (AGO1, AGO2, AGO3, AGO4) hinder translation in their target mRNAs, with AGO2 having the unique ability within its subfamily to induce RNA interference. In flies, AGO2 also triggers RNA interference in siRNA while AGO1 focuses on miRNA. What is different in flies compared to the case with mammals is that both AGO1 and AGO2 in flies can target cleavage and cause RNA interference.

Two steps in RISC assembly: RISC loading and unwinding

There are two steps involved in RISC assembly. The first step is called RISC-loading, and this is when small RNA duplexes are incorporated into Ago proteins. Prior to this step, the double-stranded siRNAs and miRNAs are converted by RNase III enzymes (Drosha and Dicer) into small RNA duplexes: siRNA duplexes and miRNA-miRNA* duplexes. In the second step, the double-stranded small RNA duplexes are separated into two strands inside the Ago protein. Of the two strands, the strand with a less stable 5’ end is kept, serving as the ‘guide strand’. The other strand, called the ‘passenger strand’, is thrown out in order to produce a functional RISC. This strand selection in which one strand is preferred over the other is referred to as the ‘asymmetric rule’.

Genome Encoded Small RNA

The Human Genome Project observed a relatively small number of protein-coding genes relative to genome size. It is believed that only five percent of the genome encodes proteins. miRNA, siRNA, and piRNA are part of the noncoding genome.

miRNA is believed to exist in hundreds of species and are identified through forward genetics by miRNA mutant isolation, bioinformation predictions based on the stem-loop, and direct cloning of small RNA. It is unclear how pre-miRNA is converted and there are studies to indicate that pri-miRNA and pre-miRNA occur separately in the nucleus and cytoplasm. dsRNA is a feature of pri-miRNA and aids in the processing into pre-miRNA. Dicer and Drosha are part of the factors required for the small RNA maturation. They are believed to function with dsRNA binding proteins which aid in the miRNA production.

Endo-siRNA play important roles in regulating genome functions in diverse species. They cleave target mRNA so that RNA-dependent RNA polymerases use the cleaved mRNA as templates to prime synthesis of secondary siRNAs. These are then loaded onto non-slicing agos to contribute to target silencing. This corresponds to the spreading of RNAi in mRNA and linked to silencing of worms.

piRNA are small RNA that also aid in the interference but focus on repetition. piRNA in mammalians are mapped uniquely in the genome and cluster to a small number of around 10 to 83 kb. Findings of the amplication of piRNA led to a ping-pong model in which it switches between Ago3 and Aubergine to create new piRN through each successive round. Different Piwi proteins conduct piRNA functions both cooperatively and independent of one another. piRNA play an important role in germ line development and the maintenance of genomic integrity. They are also involved in silencing but this is still unknown how. However, studies suggest that they regulate DNA methylation.

References

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