While working on a cell cycle lecture for the Education Resources web at Promega.com, I reread some classic papers describing classic cell-cycle experiments. Two of these papers describe the experiments by Murray and Kirschner showing that cyclin B synthesis and degradation are required for cycling in Xenopus oocyte extracts. When I took my first graduate-level cell biology course in 1989, these papers had just been published. I remember the instructors of the course being particularly excited about this work. (I also remember getting the events of Xenopus oocyte activation and fertilization mixed up on one of the tests for this course and realizing it about two minutes before I had to hand in my test, but I digress.)
Looking at these papers now with the eyes of someone who has followed cell biology for more years than I care to admit, with the eyes of an educator, and with the eyes of someone who now truly “gets” that science is an iterative process, I understand the instructors’ excitement.
These papers bring traditional cell biology, Xenopus oocyte extract protein chemistry together with molecular biology and hard-core yeast genetics to help answer a fundamental biological question: What is the nature of the “oscillator” that regulates the cell-cycle? M-phase promoting factor (MPF; at that time still called maturation promoting factor) had been isolated from oocyte extracts and shown to have kinase activity. A little reverse genetics had also shown that one of the subunits was homologous to the cdc2 gene, which encoded a kinase, from S. pombe.
In one paper, the authors showed that Xenopus oocyte extract contained proteins that behaved much like cyclins identified from other species. Then they showed that cyclin synthesis, and only cyclin synthesis, was required to induce the mitotic cycling of their extracts. They treated the extracts, which contain abundant maternal mRNA species, with a low concentration of RNAse A for a limited time, to degrade the mRNA species (but not tRNA or ribosomal rRNA, which would be essential for protein synthesis). Then they inactivated the RNase A by adding RNasin Ribonuclease Inhibitor. After the RNase was inactivated they added sea urchin cyclin B mRNA back to the extracts. Xenopus B1 and B2 cyclin mRNA were also able to drive the cell cycle. However if the mRNA contained a mutation that resulted in non-degradable cyclin B protein, the extracts would arrest in mitosis; late events such as chromosome decondensation or nuclear envelope formation did not occur.
It is clear from the paper that the collaboration between molecular biologists and cell biologists and yeast geneticists was essential for this story to come together. And, reading these papers today someone, particularly a student might be tempted to think “and that’s the whole story.” But, of course, it’s not.
Enter MicroRNAs (miRNAs). Xenopus embryos go through a developmental event called the mid-blastula transition. The first 12 cleavages after fertilization are extremely rapid, essentially just alternating S and M phases, and are driven by maternally supplied components. However, at division 13, the zygote takes over. The maternal mRNAs are degraded; zygotic transcription begins; and the cell cycles begin to include extended G1 and G2 phases. Lund and colleagues have shown that an miRNA (miR-427) is both necessary and sufficient for the deadenylation and degradation of maternal RNAs during this developmental transition.
The molecular phenomenon of antisense RNA came to light toward the end of my graduate school career (though its effects had been observed in flowers many, many years ago), and the discovery of microRNAs happened after that. But, with this new knowledge we can look back at an “old” question and add a little more understanding.
Science marches on, and new discoveries fuel our understanding of fundamental questions of biology. And it’s so cool.
- Murray AW, & Kirschner MW (1989). Cyclin synthesis drives the early embryonic cell cycle. Nature, 339 (6222), 275-80 PMID: 2566917
- Murray AW, Solomon MJ, & Kirschner MW (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature, 339 (6222), 280-6 PMID: 2566918
- Lund E, Liu M, Hartley RS, Sheets MD, & Dahlberg JE (2009). Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. RNA (New York, N.Y.), 15 (12), 2351-63 PMID: 19854872