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From the Ribosome to the Spliceosome and Back Again

2009; Elsevier BV; Volume: 285; Issue: 1 Linguagem: Inglês

10.1074/jbc.x109.080580

1083-351X

Christine Guthrie,

CRISPR and Genetic Engineering

When invited to contribute to the Reflections series, I pondered what aspects of my career I should focus on. Hoping to lay down a strong narrative arc, I have chosen to link my earliest beginnings with RNA, as a graduate student studying the ribosome, to my later adventures in what hence became known as “the RNA World,” as a tenured professor studying the spliceosome. I believe that some of my most unusual scientific insights have come from this particular path, and it is those examples I have highlighted here. Given that these articles are intentionally highly personal narratives, I want to ask at the outset for forbearance from the many members of the RNA World community whose invaluable contributions I have not been able to cite, given the length constraints, but without which my own progress would not have been possible. When I went to the University of Michigan in Ann Arbor as a freshman in 1962, I wanted to major in biochemistry. When I found out there was no such major, I toyed with English. (My mother was a writer.) I enjoyed the reading, but I resented being evaluated for my personal, obviously subjective views in writing. When my essay on Moby Dick came back covered with red ink and a large angry “C,” I decided I had had it. I would pursue science, unassailable in its objectivity! (Or so I thought.) I chose a major in zoology rather than biology, which spared me from having to memorize long lists of plant names in Latin. However, in my senior year, I was transfixed by a lecture in which it was revealed that someone had succeeded in splitting a bacterial ribosome into several pieces and putting it back together again, a biochemical tour de force. Yes, this is what I liked best of all. In a remarkable coincidence, the next year found me sitting in the office of the very man who had accomplished this feat, Masayasu Nomura. I had recently married my high school sweetheart, and he was accepted only at a single medical school, the University of Wisconsin, where I dutifully followed him. I had never thought seriously about getting a Ph.D. Only three other girls in my high school class had even gone to college. Along the way, however, my husband-to-be persuaded me to consider trying for a Master's, so I had applied to graduate school in Madison and been accepted. “I want to work on the ribosome,” I told Professor Nomura. “What grades did you get in organic chemistry?” he parried. Hearing of my 10 h of Cs (and ignoring my straight As in biology), he brusquely told me to look elsewhere. When I persevered, he told me, “Girls can't do biochemistry. They can't lift heavy rotors or spend long hours in the cold room.” “What can they do?” I asked. “Genetics,” he said. As it turned out, I would have to wait several years before a suitable ribosome genetics project was conceived. In the interim, Nomura allowed me to do some experiments analyzing how ribosomes initiated protein synthesis in vitro. This indeed entailed biochemistry. To establish that the initiator tRNA bound first to the 30S not the 70S ribosome required a complex labeling strategy utilizing radioactively (14C and tritium) and density (15N and deuterium) labeled components. The experiments were technically difficult and emotionally stressful. For example, because of the high cost of “heavy water,” every drop of D2O had to be redistilled from the growth medium for reuse. Eventually, I was able to show that the initiator tRNA was found exclusively on hybrid (heavy/light) 70S ribosomes, i.e. which had to have undergone prior initiation on 30S ribosomes. This work was published in Nature in 1968 (1Guthrie C. Nomura M. Initiation of protein synthesis: a critical test of the 30S subunit model..Nature. 1968; 219: 232-235Crossref PubMed Scopus (54) Google Scholar). After that, I was definitely ready for some genetics. During that time, the main focus of the rest of the lab had remained the elusive search for ribosomal protein structure: function relationships. Nomura believed that, when each protein was eventually purified, it could be assigned a specific function by asking what happened to ribosome performance when this single protein was omitted. Numerous male postdoctoral fellows put in their long hours in the cold room, trying to achieve “total reconstitution” of the ribosome. (The work I had heard about in Ann Arbor was, by comparison, only “partial,” i.e. a soluble protein fraction could be split off from the ribonucleoprotein (RNP) core particles in high salt and, after dialysis, etc., added back to the cores to regain activity.) For total reconstitution, a mixture of 21 proteins of the 30S ribosomal subunit was combined with purified 16S rRNA, yet despite repeated tries, no active ribosomes were produced. In what I consider to this day a grand cosmic joke, the breakthrough came when Peter Traub, a tenaciously driven German postdoctoral student, reasoned that, because cells grow at physiological temperatures, ribosome reconstitution might in fact require a temperature-dependent step. Out from the cold room he came. Indeed, when heated to 40 °C, active 30S ribosomes were reconstituted! It was a great day in the laboratory, but as more and more elegant experiments were carried out to describe the in vitro biochemical pathway, the question loomed whether this pathway had anything to do with biology. Thus was my genetics project finally born. The reasoning was that if the process of ribosomal subunit assembly in vivo was also temperature-dependent, mutants should manifest themselves as cold-sensitive (cs). Thus, I mutagenized bacteria and replica-plated them to low and high temperatures. Indeed, I found that some 30% of cs mutants in Escherichia coli were ribosomal subunit assembly-defective (sad) at low temperatures. This required me to perform double-labeling experiments (wild-type versus mutant) of cells shifted to the cold; extracts were then analyzed by sucrose gradients. In contrast to wild-type profiles containing only 30S and 50S peaks, sad mutant cells accumulated species with unusual sedimentation values. Most interestingly, in one case, the size approximated that found for in vitro reconstitution intermediates (reconstitution intermediate particles) formed without the heat incubation step (and subsequent analysis revealed a significant overlap in protein composition). Although the specific molecular blocks in my sad mutants were never determined, these experiments were taken to confirm the biological relevance of the in vitro pathway, consistent with the hypothesis that ribosome assembly requires a temperature-dependent RNA conformational rearrangement (2Guthrie C. Nashimoto H. Nomura M. Studies on the assembly of ribosomes in vivo. Cold Spring Harbor Symp..Quant. Biol. 1969; XXXIV: 69-75Crossref Scopus (35) Google Scholar). The timing could not have been better. The 1969 Cold Spring Harbor Symposium was entitled “The Mechanism of Protein Synthesis,” and the lab's combined stories were granted three back-to-back presentations in the opening session. I was completely overwhelmed to be given this rare opportunity to talk as only a third-year graduate student, and I barely slept the night before my talk. (Probably, Joan Steitz had the same problem; we shared a bathroom in the Page Motel, and I threw up repeatedly.) Nonetheless, the talk was enthusiastically received, and I was invited to give a seminar the next fall at Harvard University (to Nomura's chagrin). The ribosome was a magnet for driven scientists at the time, and competition between laboratories in the United States and Europe was fierce. Unfortunately for me, two of the fiercest competitors were Nomura and Chuck Kurland, another Madison biochemist. I had greatly enjoyed taking courses from Chuck in the newly emerging field of molecular genetics, and he had become an invaluable mentor to me as I struggled to stay free of the fallout between them, but it was not to be. Ultimately refusing to tolerate any connection between Kurland and myself, Nomura ordered me to leave the laboratory. In the early spring of 1970, my thesis committee was informed that I should receive my Ph.D. forthwith, and that was that. Adrift, I was overwhelmed by doubts that it would ever be possible to do science in a nurturing environment (Fig. 1). Indeed, this became my major goal when I finally established my own laboratory at the University of California, San Francisco, in 1973. Little did I realize at the time how profound were my “lessons from the ribosome,” both personally and scientifically. The discovery of “split genes” in 1977 was absolutely breathtaking. How could it be? Why? For those of us who had been working in the new field of “RNA processing” (first so dubbed in 1974 at a symposium in Brookhaven), this “amazing sequence arrangement” (3Chow L.T. Gelinas R.E. Broker T.R. Roberts R.J. An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA..Cell. 1977; 12: 1-8Abstract Full Text PDF PubMed Scopus (710) Google Scholar) spurred a thrilling and highly competitive search for mechanism. In 1980, Joan Steitz published the provocative idea that small nuclear ribonucleoprotein particles (snRNPs) might mediate the specificity of the reaction by virtue of complementarity between sequences at the 5′-ends of U1 small nuclear RNA (snRNA) to short sequences at the splice sites (4Lerner M.R. Boyle J.A. Mount S.M. Wolin S.L. Steitz J.A. Are snRNPs involved in splicing?.Nature. 1980; 283: 220-224Crossref PubMed Scopus (731) Google Scholar). Although this was a highly appealing paradigm, the data were circumstantial at best. At the time, I had been trying to understand the mechanism by which introns were removed from certain yeast tRNAs using a system of suppressor genetics derived from the pioneering studies of bacterial tRNAs by the group led by John Smith and Sydney Brenner at the Medical Research Council in Cambridge, England. Although still very much a novice at yeast genetics, I was captivated by the possibility of using this type of approach to understand the removal of introns from yeast mRNAs. Specifically, if Joan's hypothesis were correct, it should be possible to make splicing suppressors by introducing complementary base pair changes between a 5′-splice site and U1 and thereby prove the model genetically. I was convinced that this approach would work, but I found little support from my colleagues in the processing community. It repeatedly was pointed out to me that only a handful of yeast genes even had introns, somehow implying to them that the mechanism would be “special” and mechanistically unrelated to that in higher eukaryotes. Even more problematical was the fact that Joan had failed to detect snRNPs in Saccharomyces cerevisiae when she performed immunoprecipitations with “anti-Sm” antibodies derived from patients with the autoimmune disease systemic lupus erythematosus. The very fact that these antibodies could bring down snRNPs from all other species tested (including something called the fall armyworm) had been used to argue the high conservation of snRNP-based splicing across evolution. Ipso facto, yeast must use a different mechanism. I had a different conviction, that the failure of the antibody experiment rather reflected the divergence of the so-called Sm antigen; thus, the best approach should be to look directly for the snRNAs themselves. I based this argument on observations gleaned from ongoing analyses of bacterial ribosomes. As I described above, the efforts to get at the mechanism of translation had to date been focused almost exclusively on the roles of the ribosomal proteins. This came from a strong conceptual bias that only proteins could perform functionally sophisticated roles. Although many ribosomal proteins could be omitted in ribosome reconstitution experiments, inactivation of rRNA followed single-hit kinetics. By far the most persuasive argument for the functional importance of the RNA came from phylogenetic sequence comparisons, where it emerged that the secondary structure of bacterial 16S rRNAs was remarkably conserved. The beautiful exposition of these data by Noller and Woese in 1981 (5Noller H.F. Woese C. Secondary structure of 16S ribosomal RNA..Science. 1981; 212: 403-411Crossref PubMed Scopus (339) Google Scholar) persuaded me beyond any doubt that phylogenetics must similarly underlie the most important functional conservation among snRNAs, from yeast to mammals, yet when I announced my intentions to use phylogenetics to get at the mechanism of splicing, I was generally laughed at. Indeed, there was little to encourage me as I began my hunt for snRNAs in budding yeast. Southern analyses using cloned probes from mammalian U1, U2, and U4 RNAs were uniformly negative, forcing me to look directly for the RNAs themselves. In consultation with Joan Steitz and Alan Weiner, I chose to focus on three criteria for RNAs of interest: small size, possession of a unique 5′-cap (trimethylguanosine), and metabolic stability. From years of analyzing metabolically labeled yeast tRNAs on polyacrylamide gels, I knew that other RNAs in that size range would likely be present in very low abundance, but even when I scaled up to 50-mCi 32P labelings, there were only faint candidates, which themselves required a second dimension to be resolved from the more abundant breakdown products of rRNA. The key diagnostic was to determine which, if any, of these RNAs possessed 5′-caps. This entailed a series of enzymatic digests and subsequent analyses by thin-layer chromatography, requiring long exposures of the autoradiographs. Of course, there had been the real possibility that yeast caps might be different from the 2,2,7-trimethyl caps of mammals, but I managed to identify spots that contained at least dimethyl caps. Curiously, the number of these species was larger than I had anticipated, but that seemed the least of my problems. Cloning proved to be the most difficult step of all, and I was fortunate to attract an ambitious and talented postdoctoral student, David Tollervey, who finally succeeded. In 1983, the lab (Fig. 2) was able to publish two back-to-back papers in Cell demonstrating that yeast did indeed have snRNAs and, most importantly for subsequent genetic analysis, that these were encoded by single-copy genes, in striking contrast to the large multigene families in mammals (6Wise J.A. Tollervey D. Maloney D. Swerdlow H. Dunn E.J. Guthrie C. Yeast contains small nuclear RNAs encoded by single copy genes..Cell. 1983; 35: 743-751Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 7Tollervey D. Wise J.A. Guthrie C. A U4-like small nuclear RNA is dispensable in yeast..Cell. 1983; 35: 753-762Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Although I was feeling triumphant at this proof-of-principle stage in my program, I was increasingly troubled by the fact that sequence analysis of the first six of the cloned genes failed to reveal any obvious homology to mammalian U1–U6 snRNAs. Even harder to explain was the finding that deletion of these genes (an experiment that could be done only in yeast) produced no phenotype whatsoever. In 1985, we finally found that cells deleted for snR10 were cold-sensitive. To test the hypothesis that we were dealing with functional redundancy, we constructed multiple mutants, an increasingly tedious process given the small number of selectable markers available to track the deleted gene copies. Ultimately, we made the sextuple mutant only to find that this strain was not any more cold-sensitive than the single deletion of snR10. My talks at meetings were greeted by increasingly bored audiences, and even good friends seemed embarrassed for me, suggesting that I reconsider my research goals, but I could not let go. Where were the expected essential snRNAs, and what in the world were the functions of all these dispensable genes? While awaiting the identification of the longed-for spliceosomal snRNAs, I was fortunate to attract a graduate student who had been well schooled in yeast genetics by Beth Jones. Roy Parker brought joy to my heart by totally buying into the splicing suppressor project, so while others toiled away, screening new snRNA genes, Roy embarked on the suppressor end of things. Because the majority of known intron-containing genes were essential, he designed a reporter gene in which the intron (and short 5′-exon) was derived from actin, and the 3′-exon encoded a gene allowing cells to grow on histidinol (Hol), a histidine precursor. Thus, cells that were specifically unable to splice the reporter gene could be identified as Hol−. Because little was known about the cis-acting requirements for intron removal, Roy proceeded to mutagenize the reporter and sequence mutants. On his first try, he identified a Hol− mutant that accumulated unspliced precursor due to a mutation (G → A) at position 5 of the 5′-splice site. This work was published in Cell in 1985 (8Parker R. Guthrie C. A point mutation in the conserved hexanucleotide at the 5′ splice junction of the yeast actin intron uncouples recognition, cleavage, and ligation..Cell. 1985; 41: 107-118Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Now, if we only had a U1 snRNA… In an important breakthrough, John Abelson's lab had recently developed an in vitro splicing system and subsequently, by running the extracts over a sucrose gradient, identified an ∼40S particle containing 32P-labeled splicing intermediates, which they dubbed the “spliceosome” (9Brody E. Abelson J. The “spliceosome”: yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction..Science. 1985; 228: 963-967Crossref PubMed Scopus (227) Google Scholar). This provided an opportunity to see if any of the cloned RNAs co-migrated with this peak, experiments we carried out with Ed Brody on his sabbatical in John's lab at the California Institute of Technology, but the resolution was poor and signals low. Joan Steitz also came to do a sabbatical there, hoping to identify yeast U1 using an RNase H strategy, but this approach was equally unsuccessful. In the meantime, the competition was finally starting to heat up, and my frustration was at an all-time high. Having finally identified an essential small capped RNA, snR7, I essentially ordered the graduate student working on it to find some homology before his group meeting. Remarkably, the next day, Bruce Patterson came in smiling with the news that snR7 was none other than yeast U5: only 9 nucleotides of sequence identity, but tellingly located in a region of highly conserved secondary structure! To prove his case, he went on to clone the gene under the inducible GAL promoter, which allowed him to delete the chromosomal copy when cells were growing in galactose; in glucose, these cells accumulated unspliced actin mRNA, as predicted for an essential spliceosomal snRNA (10Patterson B. Guthrie C. An essential yeast snRNA with a U5-like domain is required for splicing..Cell. 1987; 49: 613-624Abstract Full Text PDF PubMed Scopus (98) Google Scholar). I was jubilant, thinking it would be just a short time before we could establish a 1:1 relationship with all the mammalian RNAs. My hopes were buoyed by experiments we had been performing in collaboration with Sandra Wolin, who had perfected the technique of microinjection into Xenopus oocytes. The idea was to introduce a collection of yeast RNAs into amphibian nuclei, which stockpile the Sm antigen prior to the mid-blastula transition; the in vivo assembled snRNPs should then become immunoprecipitable with mammalian anti-Sm antibodies. Gratifyingly, one of the RNAs we identified was snR7, which indeed contains a putative Sm-binding site (AAUUUUUG in a single-stranded region) (11Riedel N. Wolin S. Guthrie C. A subset of yeast snRNAs contain functional binding sites for the highly conserved Sm antigen..Science. 1987; 235: 328-331Crossref PubMed Scopus (44) Google Scholar). A second immunoprecipitable RNA was snR14, which we then predicted to also be an essential gene. When this expectation was fulfilled, we were highly optimistic that the approach was robust, although puzzled that several other immunoprecipitated RNAs were far from “small.” At about the same time, Manny Ares had made the remarkable discovery that an ∼1000-nucleotide yeast RNA contained a region with high primary sequence identity to the 5′-end of mammalian U2 snRNA. Curiously, as he continued to examine the RNA, he identified small patches of potential homology to other mammalian snRNAs, including U5. He published the sequence of what he dubbed to be a “poly-snRNA” in Cell in 1986 (12Ares Jr., M. U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs..Cell. 1986; 47: 49-59Abstract Full Text PDF PubMed Scopus (87) Google Scholar), a highly satisfying idea to those who had always maintained the conviction that yeast were “different,” yet seemingly in conflict with our identification of snR7 as U5, which was not published until 1987. Clearly, resolution of this apparent paradox would ultimately require identification of each yeast homolog as well as determination of the function of the remainder of the poly-snRNA sequences. In the meantime, I had been fascinated by the U2-like structure of the 5′-end of Manny's large spliceosomal snRNA (Lsr1). Despite some initial confusion, we soon established that this RNA was indeed represented among our cloned capped species (snR20), but which we had paid little attention to because of its suspiciously large size. It had been previously established that yeast introns shared a unique consensus, UACUAAC, upstream of the 3′-splice site. Initially, this was taken as firm evidence that yeast was “different,” as no such consensus could be found in mammalian introns. Importantly, however, it was subsequently shown that this sequence contained the site of lariat formation in the first chemical step of splicing. I was struck by the realization that Lsr1/snR20 contained a sequence with almost perfect complementarity to the so-called “TACTAAC box,” almost because it required a single nucleotide to be bulged out of an otherwise perfectly base-paired helix. I recently had been devouring papers about the self-splicing introns of the so-called Group II class because it had been shown that splicing of this class used an identical two-step mechanism involving lariat formation. That breathtaking discovery had riveted the field, as it strongly implied that, as with the ribosome, the spliceosomal snRNAs might themselves be catalytic. An elegant experiment recently had been published in which it was shown that the lariat formed on a conserved adenosine bulged out of an intramolecular helix and that this bulge was essential for catalysis. With that, I had what seemed to me the perfect rationalization for the pairing of the intronic UACUAAC with GUAGUA near the 5′-end of the yeast U2-like snRNA. The beauty of the hypothesis was that we finally had in hand the tools to perform the long-dreamed of suppressor experiment, but with U2 instead of U1. Roy Parker was just about out the door to do a postdoctoral program, but I managed to persuade him to do the test: 1) mutate UACUAAC in the actin reporter and look for cells that could no longer grow on histidinol; 2) mutate GUAGUA in a plasmid-borne copy of snR20, leaving the chromosomal copy intact; and 3) make all pairwise combinations. If the model were correct, suppression (growth on histidinol) should be observed only with the complementary pair of mutations in the snRNA and the reporter pre-mRNA. Despite the conceptual simplicity of the experiment, there were many caveats. For example, compensatory base pair analysis can work only if the sequence per se is not absolutely essential, which is a counterintuitive argument when dealing with apparently invariant sequences. Thus, a likely outcome is weak partial suppression. Moreover, mutation of an important sequence in an snRNA might confer a dominant-negative phenotype, precluding any useful interpretation. Indeed, we saw examples of both situations, keeping me on pins and needles until all combinations could be carefully analyzed and all results repeated. In the end, the data clearly supported allele-specific suppression, the genetic equivalent of atomic resolution! U2 snRNA recognizes the branch point by base pairing (Fig. 3). I was exhilarated and could not wait to see the reaction when our results were presented at the upcoming Cold Spring Harbor meeting, but the anticipated moment of triumph was dashed by the silence of the audience. Finally, a senior member of the eukaryotic splicing field sputtered, “What are we supposed to make of this? Surely this can't have anything to do with mammals!” It was emblematic of the deep suspicions held by most RNA processors, and it did not help that our primary results were genetic rather than derived from in vitro biochemistry. The paper was published in Cell in 1987 (13Parker R. Siliciano P.G. Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base-pairing with the U2-like snRNA..Cell. 1987; 49: 229-239Abstract Full Text PDF PubMed Scopus (323) Google Scholar); we proposed that the same hydrogen-bonding interactions occurred in higher eukaryotes (the GUAGUA sequence in U2 is essentially invariant) but with shorter complementarity due to the weak adherence of the sequences surrounding the branch point to the UACUAAC consensus. Nonetheless, it would be several more years and many more experiments before the relevance of our findings was widely accepted. Using a cis-competition experiment, Alan Weiner showed that UACUAAC was indeed preferred in favor of a natural branch point, and examples mounted in which the natural branch point sequence of efficiently spliced metazoan genes was indeed UACUAAC (14Zhuang Y.A. Goldstein A.M. Weiner A.M. UACUAAC is the preferred branch site for mammalian mRNA splicing..Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 2752-2756Crossref PubMed Scopus (117) Google Scholar). In 1988, we showed that all but the U2-like sequences of snR20 could be deleted without consequence and, in 1990, that the human U2 gene could in fact efficiently complement an snR20 deletion (15Shuster E.O. Guthrie C. Human U2 snRNA can function in pre-mRNA splicing in yeast..Nature. 1990; 345: 270-273Crossref PubMed Scopus (33) Google Scholar). To this day, it is unclear what function, if any, the ∼1000 nonessential nucleotides perform. A similar quandary persists about yeast U1. In 1987, we showed that snR19, a second very large capped RNA, was the homolog of U1 both structurally and, using suppressor genetics, functionally (16Siliciano P.G. Jones M.H. Guthrie C. S. cerevisiae has a U1-like snRNA with unexpected properties..Science. 1987; 237: 1484-1487Crossref PubMed Scopus (73) Google Scholar). (Notably, the first U1 suppressor experiment was actually performed in mammalian tissue culture cells by Weiner's group in 1986 (17Zhuang Y. Weiner A.M. A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation..Cell. 1986; 46: 827-835Abstract Full Text PDF PubMed Scopus (463) Google Scholar).) Similar results were reported by Michael Rosbash (18Kretzner L. Rymond B.C. Rosbash M. S. cerevisiae Ul RNA is large and has limited primary sequence homology to metazoan Ul snRNA..Cell. 1987; 50: 593-602Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Having accounted for U1, U2, and U5, it was left to identify the yeast homologs of U4 and U6, specific roles for which were unknown. In fact, we had been skeptical about looking for yeast U6 from the outset because of its unusual properties in mammals. The smallest of the snRNAs, it lacks both a trimethylguanosine cap and an Sm-binding site. In 1987, a new postdoctoral fellow came to the laboratory. David Brow had studied yeast 5S ribosomal RNPs as a graduate student and was curious to use his two-dimensional gel methodology to look at yeast snRNPs, about which little to nothing was known. In preparation for analyzing yeast cell extracts, he insisted on performing a control experiment in which he analyzed phenol-extracted RNAs, subjecting them to native gel electrophoresis in the first dimension followed by a denaturing gel in the second. He then performed a Northern blot to identify the positions of several of our cloned snRNAs. Although he had only been in the lab a short time, he came to my office very excited to announce that there was something very interesting about the mobility of snR14. “What is that?” I asked, unable to see anything unusual. He insisted that it was migrating anomalously in the second dimension, slightly off the diagonal. “So what?” I asked, wondering if David was going to turn out to be tediously hung up on minor experimental details. He pointed out that it could be an effect of unusual intramolecular structure or indicate that snR14 was associated with a second RNA that dissociated in the denaturing dimension. We had had inordinate trouble convincing ourselves of the homology between snR14 and a mammalian counterpart; it had no compelling primary sequence homology to any known snRNA. Somehow we had persuaded ourselves that it could be the U1 ortholog (prior to our analysis of snR19), and we had just submitted a manuscript to Nature to that effect. When David's subsequent control experiments convinced him that snR14 was likely associated with a second RNA, we realized that this behavior was strongly reminiscent of that of mammalian U4 and U6; Joan Steitz and Reinhard Luhrmann had previously found that these two RNAs were associated with one another. If this analogy were true, then snR14 must be U4 and the “invisible” RNA, U6. To quickly test this hypothesis, David hybridized his blot to a DNA probe from mammalian U6. Bingo! While terrifically excited by this discovery, I was simultaneously horrified that we would hav