The availability of short interfering RNAs (siRNAs) to silence gene expression has revolutionized research in molecular cell biology. But these synthetic siRNAs rely on cellular enzymes for their activity.
One classic biochemistry approach involves fishing for an enzyme in a complex mixture of proteins, where the biochemist's rod is the technique of column chromatography. Using an elegant chromatographic purification strategy, Weitzer and Martinez1 (page 222 of this issue) report catching an enzyme from human cell extract that adds a phosphate group to the 5′ end of an RNA molecule. The 'fish' turns out to be human Clp1 (hClp1), which has roles in the process of RNA interference (RNAi), in the splicing — or processing — of transfer RNAs (tRNAs), and in the formation of 3′ ends of messenger RNAs.
Normally, RNAi occurs when short interfering RNA (siRNA) molecules, 21–23 nucleotides long, silence mRNAs with complementary sequences. Silencing requires the incorporation of siRNA into a protein complex known as the RNA-induced silencing complex (RISC), which directs the siRNA to its target mRNA. The siRNA–RISC will then silence the target mRNA by either cleaving it or inhibiting its translation into a protein2. A breakthrough in the basic understanding of RNAi came with the use of synthetic siRNAs to silence target mRNAs3,4,5. For example, it became apparent that the addition of a phosphate group to the 5′ end of siRNAs was required for their incorporation into the RISC complex6,7,8. An enzyme capable of phosphorylating the 5′ end of siRNAs, however, had yet to be identified. Weitzer and Martinez searched for such a kinase enzyme in the presence of the ATP nucleotide as a phosphate-group donor, and, after fractionation of a human cell extract and eight purification steps, they isolated hClp1.
Although hClp1 was originally identified as a member of an RNA-cleavage complex involved in the formation of 3′ ends of mRNAs9, its exact function remained unknown. However, sequence analysis of hClp1 revealed a motif that is known to bind to ATP and its related nucleotide, GTP, prompting Weitzer and Martinez to consider hClp1 as the possible kinase for siRNAs. When the authors used RNAi to deplete human cells of hClp1, they found that extracts from these cells could no longer efficiently phosphorylate synthetic siRNAs. They also showed that Clp1 purified from bacterial cells had kinase activity towards siRNAs in vitro. Thus, they identified the first RNA kinase capable of phosphorylating siRNAs.
In pulling out hClp1, Weitzer and Martinez serendipitously isolated the endonuclease enzyme complex required for the removal of intervening sequences from tRNAs10. A few genes encoding tRNAs contain non-coding sequences, or introns. These must be removed to produce a mature, functional tRNA. This process of tRNA splicing is mediated by the tRNA endonuclease complex, which recognizes, cleaves and releases the introns from a tRNA molecule11.
Previously, hClp1 was identified as a member of the purified tRNA-splicing endonuclease complex, uncovering an unexpected connection between the processing of tRNAs and the formation of mRNA 3′ ends, in which hClp1 is also involved10. This work led to the suggestion that multiple endonuclease enzymes may assemble into the molecular equivalent of a Swiss Army knife, creating a complex that can cleave several different substrate RNAs10,12. So, could the RNA kinase activity of hClp1 be an additional tool within the complex, serving to phosphorylate substrates involved in tRNA splicing? This seemed possible, considering that the ligation step, necessary to stitch the tRNA halves together after endonuclease activity, requires the activity of an RNA kinase13. Weitzer and Martinez1 set out to address this question directly.
The authors pulled out either hSen2 (a known member of the tRNA endonuclease complex) or hClp1 from human cell extracts, and showed that each of these fractions was able both to release the intron by cleaving the tRNA and to phosphorylate the 3′ exon at its 5′ end. Thus, the purified complexes contained two of the three enzymatic activities required to process tRNA introns. The authors also showed that extracts from hClp1-depleted cells were severely compromised in their ability to ligate tRNA half-molecules after their cleavage by the endonuclease complex. Together, these results demonstrate a requirement for hClp1-mediated RNA phosphorylation in the tRNA-splicing pathway in human cells.
Weitzer and Martinez1 have made a big catch (Fig. 1). The ability of hClp1 to phosphorylate substrates involved in both RNAi and tRNA splicing suggests a functional link between these two fundamental processes of RNA metabolism. This, together with the previously described connection between pathways involved in tRNA splicing and mRNA 3′-end formation10, raises a number of questions. Unlike synthetic siRNAs, processing of siRNAs in vivo results in siRNA molecules that contain a phosphate group at their 5′ end. Does hClp1 contribute to the natural RNAi pathway at all? Weitzer and Martinez demonstrate that hClp1 can phosphorylate several other types of RNA, in addition to siRNA and tRNA. So can the hClp1-containing complex moonlight for other RNA-processing pathways? What is the function of hClp1 in the formation of mRNA 3′ ends? Here, Weitzer and Martinez suggest that hClp1 might maintain a phosphate group on the 5′ end of cleavage products, allowing for efficient catalysis and proper termination of gene transcription by the enzyme RNA polymerase II. Finally, what is the underlying reason for organizing this group of RNA-processing complexes together? Perhaps linking RNAi, tRNA splicing and the formation of mRNA 3′ ends allows the cell to modulate RNA metabolism in response to ever-changing growth conditions.
Fishing trips to the cold room can yield fundamental insights such as that described by Weitzer and Martinez. We can expect future catches to include the tRNA-splicing ligase, as well as other proteins involved in the RNAi pathway and mRNA 3′-end formation.
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Orphanet Journal of Rare Diseases (2011)