SNURPs and Serendipity
Transcript of Part 1: SNURPs and Serendipity
00:00:29.29 Hi, my name is Joan Steitz. I'm a professor at Yale. 00:00:33.20 And what I'd like to tell you about today is the importance of 00:00:37.10 serendipity in the discovery process in science. 00:00:41.22 The story I'm going to be telling you has to do with SNURPs 00:00:45.18 which are little particles inside all of our cells that are the building blocks of the spliceosome 00:00:53.01 which assembles on pre-messenger RNAs to remove the introns 00:00:57.05 and make the messenger RNA that can be translated by the ribosome. 00:01:01.10 And these little SNURPs, as you see in this slide, 00:01:04.14 have both RNA molecules and protein molecules in them. 00:01:09.09 The other way in which this story is unusual is that it's definitely not a bench to bedside story. 00:01:17.24 Instead it's very much a bedside to bench story 00:01:21.15 where we made use of something that physicians had known about for a long time 00:01:26.00 in order to make discoveries about the basic workings of cells. 00:01:31.11 So the story starts in the early 1970s and at that time of course we knew about the central dogma. 00:01:39.25 We knew that DNA made RNA, and RNA made protein. 00:01:44.00 What we didn't know about was introns or splicing. 00:01:48.04 People had worked out many of the steps in gene expression in bacterial cells 00:01:54.07 and most people thought that higher cells would be sort of similar. 00:01:59.07 Perhaps a little bit more complicated but no new principles. 00:02:03.17 How wrong we were! 00:02:05.14 What we did know in the early 1970s 00:02:09.03 was that there were several things that were peculiar about higher cells. 00:02:13.19 We knew that they had way too much DNA, a hundred to a thousand times more than 00:02:19.02 we thought they would need in order to encode the number of genes that we thought we had. 00:02:24.08 And the other things was that RNA, 00:02:26.14 lots of it got made in the nucleus but about ninety percent of it 00:02:30.21 got degraded and only ten percent of it ever made it out to the cytoplasm to be a messenger RNA. 00:02:37.19 So, what was going on? 00:02:39.16 And as a young assistant professor at Yale, 00:02:42.02 I had studied RNA structure and function in bacteria and bacteriophages 00:02:46.21 and I decided I wanted to take on this problem on my first sabbatical leave. 00:02:52.09 We knew that there were RNA binding proteins inside mammalian cell nuclei 00:02:57.10 that immediately bound to the newly synthesized messenger RNA and I thought if I had 00:03:03.28 antibodies against those proteins, I could use them as tools and maybe this would help me 00:03:09.20 figure out how the cell decides which molecules to degrade and which molecules to send out 00:03:16.06 to the cytoplasm as messenger RNAs. 00:03:19.29 So, for half a year, I tried to make antibodies 00:03:23.20 against these nuclear RNA binding proteins but this was without success 00:03:28.07 because these are very highly conserved proteins and therefore very non-immunogenic. 00:03:33.17 So I gave up and did something else for the rest of my sabbatical. 00:03:37.10 But it was the year 1977 in the summer that introns were discovered. 00:03:43.05 Evidence came together from laboratories from all over the world 00:03:46.26 that conclusively convinced everyone that our genes, in fact, were not like bacterial genes 00:03:53.26 but contained segments of nonsense, the so-called introns. 00:03:58.03 And that these introns were then spliced out once the entire gene was made into pre-messenger RNA 00:04:05.03 and that was what enabled the cell to send a fully formed message 00:04:10.11 `to the cytoplasm to be translated by the ribosomes. 00:04:14.01 So the question then was: what is the cellular machinery that very precisely recognizes 00:04:20.07 the ends of the introns and allows the splicing process to be exact? 00:04:27.07 When I went back to my lab in the fall of 1977 everybody wanted to work on this process. 00:04:32.11 We were an RNA lab, but frankly we were pretty clueless about what we should do next. 00:04:38.05 And then came the first piece of serendipity, in January of 1978 a new issue of the journal Nature 00:04:45.13 arrived in the lab with this rather complicated title, but the important point 00:04:51.10 was the underlined sentence here which says that patients with MCTD, 00:04:56.19 mixed connective tissue disease, 00:04:58.26 have antibodies against nuclear RNA protein complexes. 00:05:03.28 And this caught my attention because while I was trying to make antibodies and failing 00:05:09.21 several people had said that they thought they'd heard of some diseases where 00:05:14.06 patients made auto-antibodies, antibodies against their own cellular components, 00:05:19.10 that reacted with nuclear RNA protein complexes. 00:05:24.10 At that time I had in the lab a new M.D./Ph.D. student, Michael Lerner. 00:05:28.27 He was fresh from his medical school courses and I said, 00:05:32.04 "Michael, do you know anybody here at Yale Medical School 00:05:35.15 who might have patients with these diseases?" And he said, "Sure, I'll go see Harden." 00:05:40.20 Harden was right across the street in the rheumotology section 00:05:44.07 and that very afternoon Michael came back 00:05:47.10 with several small vials of serum from patients with suspected auto-antibodies. 00:05:53.12 Now, the sobering part of this story is that if this were to happen today, 00:05:58.03 one would have to spend weeks if not months filling out human investigation forms, 00:06:05.05 getting it through committees in order to use even just a few milliliters of serum 00:06:11.23 `from a patient in an experiment. 00:06:13.25 So, this probably never would have happened. 00:06:15.25 But in any case, Michael started working with these sera and the idea was that the auto-antibodies 00:06:21.16 would then recognize the cellular component and we would use 00:06:26.04 them as tools to both characterize the, to purify and to characterize the function 00:06:33.24 of the target of the auto-antibodies. 00:06:36.17 And here, what you see in this picture is the idea that maybe RNA was in there and protein as well. 00:06:42.24 And we now know that those were SNURPs and that patients with lupus 00:06:47.05 and other autoimmune diseases often make antibodies against SNURPs. 00:06:51.17 However, the going was rough. As Michael purified, the cellular component kept disappearing. 00:06:59.19 We now know because ribonuclease 00:07:01.22 was in the preparations and kept eating away at the RNA component. 00:07:07.01 And then the second piece of serendipity happened. 00:07:10.07 And this was a seminar visit by Joan Brugee who is now professor at Harvard Medical School. 00:07:17.10 And she came and talked about a new reagent called Protein A that she was using 00:07:24.24 to capture immune complexes made with lysates of virus infected cells 00:07:31.19 and antibodies against those proteins. 00:07:34.14 And the advantage of that was it could be done very fast. 00:07:38.15 And you could also look at radioactively labeled cell lysates. 00:07:43.00 And what we see here is that, on the very far side, is the total RNAs in the nuclear extract 00:07:52.20 of HeLa cells labeled with P32. 00:07:54.22 Going from the very small ones at the bottom, about seventy nucleotides long, 00:07:59.07 up to U2, which is about two hundred nucleotides long. 00:08:02.16 Michael's own serum, which was used 00:08:04.24 as a control in the next lane immunoprecipitated no RNAs, but what you see 00:08:10.18 on the right-hand side here, is the pattern that he obtained with various 00:08:16.06 different auto-antibodies from various patients. And for the first time, we could see what the 00:08:22.05 RNAs were and then we could also use this to find out what the proteins were 00:08:27.22 in these little particles. So, what we now know, 00:08:31.07 is that these very highly conserved and very abundant targets of the auto-antibodies are, 00:08:37.04 in fact, the SNURPs that, by base pairing, 00:08:40.19 recognize specific sequences in the pre-messenger RNA, 00:08:44.29 gather together with a lot of other factors, to form the spliceosome that carries 00:08:50.09 out the two-step reaction that's involved in pre-messenger RNA splicing 00:08:56.14 to give, finally, the messenger RNA that can be translated into protein. 00:09:04.10 This isn't just test tube fiction, here you see a beautiful picture from Anne Byer's lab 00:09:08.17 in Virginia, showing DNA with nascent RNA transcripts coming off it, 00:09:14.16 particles building up at the five-prime and three-prime ends of an intron, 00:09:19.06 coalescing to form a spliceosome, with the intron looped out, which will then 00:09:24.19 shortly be removed by the actual chemical process of splicing. 00:09:30.11 So, what I've told you is about how serendipity 00:09:34.15 and being interested in RNA-protein complexes 00:09:37.23 in mammalian cells led to the discovery of SNURPs, which contain snRNAs and proteins, 00:09:44.05 and how that process is essential for making messenger RNA. 00:09:49.14 So, these snRNAs are, in fact, the first non-coding RNAs that we know to be important 00:09:56.17 in the regulation of gene expression. 00:09:58.20 Today we have microRNAs, lots, and lots of others. 00:10:01.26 And also, as we've understood about the spicing process, 00:10:05.11 we know that splicing is the reason why 00:10:09.17 we can have the same number of genes in our genome as the fruit fly Drosophila 00:10:15.09 and yet be more complicated. And this is because we do splicing in alternative ways, 00:10:21.09 therefore, making the most out of every gene, making multiple products from every gene.