Formation of RISC and RNA interference

Three different classes of RNA are involved in RNA interference (RNAi): microRNAs (miRNAs)¹ were first described in C. elegans to exhibit regulatory functions via complementary binding² while short interfering RNAs (siRNA)¹ were analyzed five years afterwards³. Here, the great regulatory potential of dsRNA compared to single-stranded RNA was analyzed. The third group of RNA are Piwi-interacting RNAs (piRNA)^4,5 that have a distinct interference mechanism than the other two. For mi- and siRNA the fundamental mechanism is the same: they both interfere with RNAs and require a certain degree of sequence complementarity¹. For these two RNA classes most of the processing pathway is very similar:

Both mi- and siRNA derive from longer dsRNA precursors^1,6 that get processed to shorter fragments. During processing one strand is aborted and only a single strand is loaded onto the so called RNA-induced silencing complex (RISC)⁶. The RISC comprises an endonuclease⁷ that exhibits the – mostly – inhibitory function of RNA interference. RNA interference thus is a specific type of RNA degradation and shares several features with other RNA degrading pathways (see section 1.3).

Despite important similarities all pathways differ significantly: miRNAs are usually transcribed by the RNA polymerase II⁸ or III⁹ (Figure 1.2 A). These products are called primary miRNAs (pri-miRNA)⁶ and are very often co-transcribed with the coding regions nearby or derive from mono- or polycistronic clusters^10,11. In fact they often are indeed introns⁶. These precursors are further processed by the so called microprocessor comprising Drosha¹², an RNase III enzyme¹³, Pasha¹², a double-stranded RNA binding protein, or in humans DGCR8 (DiGeorge syndrome critical region 8)¹⁴. Besides their function in RNAi Drosha is involved in neurogenesis via suppression of the transcription factor NFIB¹⁵ and DGCR8 can also act independently and recruit the exosome to degrade dsRNAs¹⁶. While DGCR8/Pasha¹⁷ is crucial

for accurate positioning of the RNA within the complex^14,18, Drosha harbors the catalytic unit that cleaves the RNA¹³. The product forms stem loop RNAs (pre-miRNA) which are exported via exportin 5¹⁹ from the nucleus to the cytoplasm where another endoribonuclease Dicer (Dcr) cleaves off the loop to leave a dsRNA²⁰ (Figure 1.2 B). One of the RNA strands (called miRNA) is loaded onto an Argonaute protein (Ago) that forms the functional and mature RISC^6,20 (Figure 1.2 D). This transient complex is called the RISC loading complex (RLC) and comprises a dsRBP, Dicer, Ago and the respective RNA^21,22 (Figure 1.2 C). Potentially certain factors like TAF11 support tetramerization of RLC components which might enhance loading activity²³. The other strand (miRNA*) is usually degraded. However, several cases are known where the miRNA* remains intact and exhibits a specific function, e.g. miR-9*²⁴ and miR-21*²⁵. Though most miRNAs are processed as described above, some have different origins, processing and function: Specific miRNAs derive from the spliceosome where an intron forms a miRNA called mirtron^26,27. In these cases no processing with Drosha is required, instead the RNA can directly be cleaved by Dcr²⁶ and is loaded onto RISC. Similarly to siRNAs miRNAs may also derive from viruses²⁸.

MiRISC binds sequence-dependently to mRNAs and inhibits protein translation^6,29 (Figure 1.2 E). MiRNAs act post-transcriptionally on both translation intiation by blocking proper ribosome assembly³⁰ or other mechamisms³¹ and translation elongation³². Therefore, usually only a partial sequence complementarity^1,33 within the seed region (2-8 nt) and the 3’-UTR of the RNA is necessary allowing miRISC to silence multiple RNAs at once³⁴ which often derive from one gene cluster. In case of perfect matching miRNA miRISC acts like siRISC and leads to mRNA decay³⁵. MiRISC thus does not only inhibit specific functions, it can also enhance degradation of mRNAs.

Figure 1.2: Schematic pathway of RNA interference. A) Pri-miRNA are processed by the microprocessor complex comprising Drosha and DGCR8 or Pasha. The pre-miRNA and endgoneous siRNA get exported from the nucleus via exportin-5. B) Pre-miRNA and both endo- and exogenous siRNA are bound by Dicer and a dsRBP and are processed into smaller double-stranded fragments (19-23 nt). C) Upon binding of an Argonaute protein the RISC loading complex (RLC) forms. Strand selection occurs based on thermodynamic stability of the RNA duplex termini. D) After degradation of the passenger strand the guide strand is loaded onto Ago to form the mature RISC. E) The RISC binds sequence-specifically to its substrates and exhibits various functions. Green arrows indicate a positive enhancement.

In contrast to miRNAs siRNAs can be both endogenous or exogenous^36,37 (Figure 1.2 A). They either derive from transposons, RNA hairpins, pseudogenes or viral RNA^6,38,39. Like miRNAs they are processed form long dsRNA precursors but in contrast to miRNA precursors they are perfect matching⁴⁰. In contrast miRNAs may contain not perfectly base-paired sequences, mismatches and loops. SiRNAs are directly processed in the cytoplasm by Dcr and

the mature siRNA is 19-21 nt long, comprises a 5’ phosphate and a 2 nt 3’ overhang^6,40, both necessary for Ago binding⁷. As for miRNA one strand of the double-stranded siRNA precursor gets degraded (passenger strand)⁴¹ while the guide strand is loaded onto Ago to form the RISC (Figure 1.2 C, D). In general strand selection depends on the thermodynamic stability of the siRNA duplex⁴²: The strand with the less stable 5’ end is maintained and part of the functional RISC. However, in case of lacking clear thermodynamic asymmetry both strands might get incorporated into a functional RISC⁴². Like the miRISC the siRISC recognizes its targets via sequence complementarity. Upon binding of the respective substrate Ago cleaves the RNA between nucleotides 10 and 11 of the guide siRNA strand⁴³, the two products get released in a specific order⁴⁴ and are then further degraded by other pathways (see section 1.3).

Both mi- and siRNAs seem to play important roles in cancer development^11,24 as well as other diseases²⁵ and could serve as potential drugs for various diseases. Especially for diseases based on misregulated genes, e.g. cancer or metabolic diseases, and for viral defense siRNAs could be a potential tool⁴⁵. In Drosophila for example siRNAs have been proven to be an effective anti-viral molecule⁴⁶.

The third class of RNAs are piRNAs which differ in many aspects from the previously mentioned RNA classes: Usually they derive from heterochromatin clusters⁴⁷ and prevent transcription of transposons^47,48 which could have a negative impact e.g. on germ lines and disrupt genes. PiRNAs do not require processing by Dcr and are slightly longer than mi- or siRNAs (26-35 nt)⁴⁹.