Ambion: You are considered to be both a pioneer and leader in the study of small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs). Can you tell us how you first became interested in this area?

Dr. Steitz: It is a long story and has been written about in two places: a 1988 Scientific American paper (Steitz JA (1988) Snurps. Sci Am 255(5): 56-63) and the Ergito website. But let me tell you briefly. It was very serendipitous. While on sabbatical, somebody told me about these antibodies, auto antibodies. But I had no way of finding the clinicians who would have access to the patients with the auto antibodies at that point. Then, when I came back to Yale, there was a paper in Nature that reminded of these auto antibodies. At that point I had a MD/PhD student in the lab who knew the people in rheumatology. He was able to get sera from such patients and we started working with it. These were the antibodies against "snurps", which ultimately led to their discovery.

Ambion: I once read that you were initially interested in attending medical school. What made you decide to pursue your Ph.D. instead?

Dr. Steitz: I got a summer job working in a lab and for the first time was given my own project and told to do whatever I could. I had worked in labs previously but only as a technician for somebody else. Having my own project and my own goals was so entrancing, I decided that I really wanted to do a Ph.D. instead.

Ambion: Can you tell us a little about being James Watson’s first female graduate student?

Dr. Steitz: First of all, I didn’t know I was the first female graduate student until several months after I joined the lab. There were so few women in the field in at that time - say 1 in 10 to 12 - and there weren’t that many women who were graduate students in this new field of molecular biology. By the time I realized it, we were fairly well into it. He was an excellent mentor, very supportive and very non-discriminatory. I owe him an awful lot in terms of serving as an inspiration and setting paradigms on how one should go about doing things - focusing on the important questions in science, how to organize and run a lab... He was very good at running a lab.

Ambion: Can you tell us what led to the recent discovery of a second spliceosome?

Dr. Steitz: This is very interesting and I have some guilt feelings about it. Databases began to show the presence of introns with splice sites that did not conform to the consensus for the classical major splicing machinery. At a very early stage, Richard Pagent came up with the idea that some small RNAs discovered by a graduate student in my lab in 1988 might be involved in the “second spliceosome”. He asked me to consider submitting a paper to PNAS. I did this and got it reviewed. But the theory seemed so preposterous and so “out of the box thinking”, and without more proof I told him, “No, sorry, this cannot be published.” About 4 or 5 years later, when there was more evidence from the database, we became very heavily involved in doing the experiments that actually demonstrated a second spliceosome. His (Pagent’s) lab has continued to make very nice contributions in parallel; again proving that there is a second spliceosome and characterizing it. I have always felt guilty that when he tried to push this idea when it was really pioneering, I stomped on it instead of saying, “That is a cool idea!” It did turn out to be right. I mean, it is true that there wasn’t much evidence, but I probably could have had the paper published for him. But I didn’t because it just seemed so “way out”.

Ambion: The rare introns that this spliceosome removes have consensus sequences that are believed to be at least a billion years old. What is the current speculation as to the evolutionary origin of this novel spliceosome? And why have the few introns it excises been maintained?

Dr. Steitz: <laughing> Well, gee, it sounds like you are writing my grant application! Current speculations about the evolutionary origin have really been set out by Burge, Sharp and Pagent in a couple of articles. I think the most intriguing ideas are that the two spliceosomes have evolved separate cell lineages and those cell lineages fused, and then you had genes that were spliced by one versus the other. Now we know that there are more and more of these introns - the number is now up to about 1 in 300 in the human genome – and that several additional genes that have more than one of these introns. This again suggests that they may have arrived in the same cell together, which goes back to this idea of there being two lineages with two different splicing systems and some sort of fusion brought them together. But, like all evolutionary arguments, it is never going to get proven. Still, I think it is very interesting.

Ambion: Your lab has another recent finding - small nucleolar RNAs (snoRNAs). Can you tell us a little about them? I read where you recently developed a coupled in vitro splicing/snoRNA-processing system. Can you tell us how you are using that to study snoRNAs?

Dr. Steitz: Yes, this is really cool. The really intriguing part is that in our cells, vertebrate cells, all snoRNAs are encoded in the introns of protein coding genes and so their synthesis, in a sense, has to be coupled with those transcripts. We know, for instance, that there are brain specific snoRNAs. This is because there are brain specific proteins and brain specific transcripts that arise because of brain specific promoters. I think one of the really exciting things that is happening in gene expression at the moment is trying to understand how the different steps (such as transcription, translation and export) – which have previously been more-or-less worked on separately – how they are all talking to each other and what the interconnections are. There have been a number of reviews on the links between transcription and the various steps in processing – processing and export, and processing and translation. Some of the things (e.g. proteins, RNAs) that get on an mRNA in the nucleus stay on it and go out into the cytoplasm and are still there when it is translated. The interesting thing about the snoRNAs is that their processing and their release from the introns is clearly coupled to splicing. We are using our in vitro system to figure out what exactly is the molecular mechanism – how this is happening. What’s clear is that you have to get to a certain stage in the splicing reaction before the proteins that define the snoRNAs actually get onto their binding site. So something is happening there, either in terms of the splicing machinery recruiting the proteins or changing the structure of the snoRNA so that it can bind the proteins. We are not sure which yet. There are very intricate interplays there, which is just yet another manifestation of splicing being at “the center” and its talking to things both upstream and downstream of gene expression. Another aspect of this is the business of the exon-junction complex. After splicing, this big complex of proteins ends up sitting on the RNA just upstream of where the intron was, and that goes through the nuclear pore, apparently with the message, and then talks to things in the cytoplasm. This is all just absolutely remarkable.

Ambion: Your study of mRNA stability has led to insight into mRNA export from the nucleus and the discovery of certain SR proteins (general splicing factors). Can you tell us about these proteins and your use of cell-permeable peptides designed to help study these?

Dr. Steitz: SR proteins and export is another example of the links between splicing and another step, in this case export. We should soon have a paper about SR proteins, basically saying that they serve as adaptor molecules for getting mRNAs out of the nucleus in the same way. There’s a transport protein called TAP that pretty much everybody accepts in yeast and vertebrate cells as the major exporter of mRNAs. This protein is known to interact with nuclear pores – part of the nuclear pore. On the other hand, it also interacts with RNA binding proteins. The message binds these proteins, now called adaptor proteins, and then the adaptors interact with things like TAP. TAP interacts with the nuclear pore and that’s how messages get out. Previously an adaptor called REF has been characterized as interacting with mRNA, both spliced and unspliced, and interacting with TAP. It appears that SR proteins bind to the same site on TAP – that they are just another kind of adaptor. How this all fits together, who's dominant over whom, and how many of these interactions you have to have to get a message out, I find to be very difficult and challenging problems, and that is sort of where everybody is.

Ambion: I read that you were planning on using array analysis to help in your study of the SR proteins. Can you tell us about this approach?

Dr. Steitz: We are using array analysis to try to sort out protein-protein interactions that are essential for the export of particular mRNAs. The idea is to interfere with these adaptor/receptor interactions necessary for getting mRNAs out, and then ask, “Which RNAs don’t get out?” The use of cell-permeable peptides is part of that approach because that’s effective in blocking protein-protein interactions in the vast majority of cells.

Ambion: You have so many different projects currently being pursued in your laboratory. How do you decide what to pursue (i.e., one project leads into another, student proposals, literature review sparks idea, etc.)?

Dr. Steitz: Yes! All of the above: one project leads into another, post-doc proposals, literature review sparks an idea...

Ambion: How many people do you have currently working in your lab? (Breakdown? i.e., graduate students, post-docs, etc.?)

Dr. Steitz: There are just over 20 total: 9 post-docs (including one gentleman who is a resident in Ob/Gyn), 4 graduate students, a student from Germany who is working on her diploma, 3 undergrads and, well, a good assortment.

Ambion: As a professor of molecular biophysics and biochemistry at Yale, a Howard Hughes Investigator, editorial board member of Genes and Development, etc., do you still have time to do any bench work?

Dr. Steitz: Only when I am on leave, so, theoretically, every seven years. The last time I was on sabbatical, which was right after I finished being chair of the department, I went to Australia for a couple of months and did work in the lab. It was great fun.

Ambion: Which of your many activities do you find most rewarding?

Dr. Steitz: The one that was really lots of fun, but also lots of work – and I actually just got rid of it because it was time for somebody else to do it – was being head of the Jane Coffin Childs Memorial Fund for Medical Research. I just handed this over to Randy Scheckman as of last July because I had done it for 11 years. It was really fun to interact with good post docs and to read good post doc applications and interact with a wonderful family foundation that is interested in supporting the future of science by supporting young people who are doing their post docs.

Ambion: How do you believe being a woman has affected your career as a molecular biologist?

Dr. Steitz: Well, I think mostly positively. I still think that because there are fewer women, especially as you sort of advance up the ladder in a field, that women tend to get noticed a little bit more. And if what you are doing is good stuff, that, plus the notice factor, I think helps.

Ambion: What advice would you give to young women scientists today?

Dr. Steitz: You have to love what you are doing. But what is wonderful now, that wasn’t the case when I was starting out, is that there are women; there are women who will be your peers and there are women who are older than you who are potential mentors and advisors. It was a pretty lonely place when I started out. So the key is to take advantage of the resources that are available and pursue your dreams.

Do you believe they face challenges unique to women?

Dr. Steitz: Yes, just because women in general get stuck with more of the family responsibilities than men do, and it just makes it that much harder. But that is true of everything women do, I think.

Ambion: What is next for Joan Steitz?

Dr. Steitz: Well, I certainly enjoy being able to think about science, more than was possible when I was doing a substantial amount of administrative work. So more administration is certainly not next. There are interesting things that need to be done in terms of efforts at universities and efforts on the national level - in science education and encouraging women to go into science. There are lots of things that need to be done, so there will be more of that.