micro-RNA: Ramifications

micro-RNA: Part Two
Ramifications

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Since very little is known about the μ-RNA's, we must approach the subject in a way very much differently than other subjects are presented in this website. Indeed, this might easily be construed to be a lesson in exploration - how to find new facts - the main process that is called "science." All we can do is use our "reasoning powers" to push forward, and perhaps even arrive at testable hypotheses. So let us begin reasoning! What will be done is to pretend that you are sitting at a table in a coffeeshop with three scientists who have all just read the article in Science News and are discussing it. The three scientists are:

Dr. Scarlet Hara, who is a young professor, and, as youth is so wont to do, is eager to learn the minutia of techniques and very small facts - usually without the wisdom that is acquired by more senior people.purple
Dr. Tony Indigo, the wizened elder stateman of the group, and interjecting "big picture" ideas.
Dr. Amy Black, who is 35ish and is just beginning to become wizened, but, alas, she and Scarlet have more than a trace of personality conflict
You!

THE CONVERSATION OVER LUNCH.
Amy: Say, did any of you see the article on micro-RNA's in Science News (ref)? These are very small RNA's that people previously thought were junk. But maybe not. If indeed the μ-RNA's are truly control elements, there must be several species of them, and the code within any one species must be fairly well conserved - certainly not random and free to mutate at will without any consequences to the organism.
Scarlet: Ahhh, before we jump too far ahead of ourselves I think that we need to put all this into context. Wouldn't you think μ-RNA's might find themselves in the general subject are of biological controls just like genes are controllers? In fact, aren't all of the controls of a cell encoded in the genes?
Amy: Scarlet! How can you say that? I would have expected you of all people to know that not everything in a cell is designed by genes and their protein products. I am aghast! Take membranes for just a trivial example.
Tony: Calm down, Amy. Let's not get sarcastic. We don't need a cat-fight here. Remember, we have a guest sitting here with us. Membranes are hardly trivial. And I might add cell walls also - both of bacteria and plants, to your list of cell parts that are not genetically programmed.
Amy: You're right, Prof. Indigo. Sorry, Scarlet. I guess all of us have holes in our knowledge. Let me try to explain it to you by giving you a classic experiment that has been known for at least 40 years.
If you grow some E.coli in 12% sucrose (that's an isotonic solution for E.coli), you can dissolve off their cell walls with lysozyme. Because of its being isotonic, the membranes don't burst, and the cells change from being little sausage shapes into spheres that are imaginatively called spheroplasts. These spheroplasts continue happily growing and dividing in the presence of the sucrose and lysozyme. But look what happens when you remove the lysozyme by some biochemical trick: the spheroplast population just continues growing as spheroplasts! Not as sausages. The spheroplasts simply cannot make new cell wall. Yes, they are chock full of the building blocks for making cell wall, but they just don't come together. It was soon found that if you were to stir in the merest of pinches of purified E.coli cell wall to the spheroplast culture, within seconds the whole culture goes frantically about making cell walls and in a minute or two there are no spheroplasts left - all are now sausages! It's almost like adding a seed crystal to a super-cooled solution of salt or sugar. It almost explosively forms crystalline precipitates. Just needed a little starter material - sort of like showing the cells how to construct cell walls from all the building blocks they have already to go. So you see, Scarlet, the E.coli cell doesn't have the genes for the general plan of its cell wall. And the same is the case for all cells and their membranes. These are cases where the "plan" is stored in the stuff itself. Membranes beget membranes; cell walls beget cell walls.
You: Hey, that gives me an idea for a project! I am so excited. Wonder what would happen if I added cell walls isolated from Bacillus to E.coli spheroplasts. Are all the right building blocks there? Or maybe Bacillus is too unrelated to E.coli. Perhaps I would be limited to some Gram-negative cell walls for "seed crystals."
Tony: See, what did I tell you. We have great students! But I think we have strayed a bit. Let's get back to DNA-based genetics. Why don't I start jotting down things we know about micro-RNA's. Maybe a picture will come into focus as to where we should head our research. Okay, I'm ready. Who's got something for my list?
Scarlet: Well, Dr. Indigo, if they are truly DNA transcripts, we might that they have a conserved genetic code - any given type of μ-RNA would all have the same sequence. And therefore we should expect to find their genes scattered somewhere on the chromosomes of their eukaryotic hosts. But where?
Yes--, and do these genes take up much space as if they were either numbered in the thousands of species, or highly redundant as are the tRNA genes in the nucleolar organizer region...
Amy: Yes, about 10,000 copies of each "t-gene" more or less.
Scarlet: But where are these μ-RNA genes hiding on the chromosome? Unlike in T4 phage where somewhere like 90% of the chromosome can be accounted for in RNA-DNA hybridization studies, eukaryotic DNA seems to have a very low percentage of DNA complementary to large molecules of RNA. Does this mean that most of eukaryotic DNA is non-coding, or that the RNA isolated for hybridization studies was only from one of many, many different portions of the cell or organism's cycle. So my question stands: where are these μ-RNA genes located on the chromosomes?
You: Introns? My teacher was just talking about introns - whatever happens to the snipped-out introns?
Tony: Hmmm, I think you might have something there. Maybe the origin of some μ-RNA's is that they are excised introns. I believe that there are several bits of reasoning in support of this candidacy. Hmmm. Apparently, a minimum of 50% of eukaryotic cistrons consists of introns. This would be a shocker of apparent inefficiency if all these stretches of DNA and RNA were only to be thrown away. A huge portion of transcript RNA is clipped out and seemingly thrown away and digested. I say 'seemingly' because that discarding is not proven - just assumed. Let me scribble this down - good stuff!
Amy: I was just reading somewhere something supporting the notion that these excised introns may serve a useful purpose. It was shown by a wonderful experiment. Two strains of SV40 (simian virus 40) were genetically engineered to contain the coding sequence for some human protein. In one strain, the inserted piece was normal human DNA with all the introns in it, and the other strain contained an inserted piece with all the introns excised out. These two strains were then used to infect a human cell culture. SV40 goes through two stages of infection/unwrapping. In the first it passes into the cytoplasm, and in the second it passes into the nucleus. Only the first strain made the human protein. Of course, an alternative value of the intron excision may only have to do with the linear transport of the transcripts out of the nuclear membrane and into the cytoplasm.
Scarlet: Black, I might add another interesting factlet: the excision of introns is far from a random event: first one is excised and the site ligated, and then the next one is excised and ligated, and so on. And it always proceeds in the same order for a given gene. This is highly reminiscent of the "clock" mechanism in T4-phage that governs the passage of time in the infection cycle separating the cycle into early, middle and late phases. T4 does this by placing a different gene for one of the components of transcriptase at the end of each of the phase's stretch of DNA. When transcriptase-1 reaches that point, the gene is expressed as a protein that replaces one portion of transcriptase-1 converting it to transcriptase-2, which can only begin reading an operator at the beginning of the next phase's portion of DNA. So the new transcriptase-2 starts reading there at operator-2 (but not back at operator-1). Eventually it reaches another gene, which expresses a factor that converts transcriptase-2 to t'ase-3. Thus this phage uses as its clockwork escapement the time it takes transcription of a long, poly-cistronic gene sequence to be read.
Tony: That sounds good. Let me get those thoughts down here on the list. You are saying that with the sequencial exision and ligation of introns, it sure looks as if the first intron fragment may be needed to do the next intron's exision, and so on. Thus the intron fragments (similar in size to μ-RNA's) may have at least one control function. Of course, to begin the exision of the first intron would require a "helper-RNA" from elsewhere.
Amy: And that would mean that the introns must be highly conserved sequences. Are they?
Scarlet: They are indeed! And they are indeed DNA transcripts.
Tony: Slow down: I am having trouble getting all this down. I am really happy to have you young hotshots here - you can remember all the details. They escape an old guy like me.
Amy: I would like to ask a series of questions dealing with the physiology of the transport of the transcripts through the nuclear membrane. As soon as one thinks of a long piece of linear RNA passing through a pore, one has a primative sort of production line - at least one station on a production line. As the linear piece continues to pass by, the pore enzymes do their snips and weldings - or ligations. But first the RNA must first be "capped" (as a needle on thread) just to be able to begin passing through the pore. Then the production line does its work. But the speed of the "production" must somehow be regulated. Also what must be regulated and balanced by feedback from the outside cytoplasm must be which transcripts are preferred to be capped and threaded through the pores.
Tony: No, we are not at a loss for possible locations of μ-RNA genes or of their functions. We've got more items on this list that I thought we would be able to think of. So now to the nitty-gritty: what sorts of experiments can we propose to see if any of our speculations are correct. If we can hone our speculations into experimental questions, we finally have hypotheses!
Oh, another thought just came to me: why don't prokaryotes show evidence of having introns? Some evolutionary microbiologists are known to say that microbes started out inefficient, and rapidly streamlined their various machinations for simplicity and rapidity of function so that if the environment favored them, they would be johnny-on-the-spot and be able to grow their populations quickly. Most eukaryotes don't seem to place that much emphasis on rapidity of growth - there is simply no way that they can beat the microbes to the punch. But eukaryotes do things better by specializing for being able to eat only certain things, or for poisoning or eating competitors.
You: And then come the guard dog bacteria I've been learning about, which do kill competitors - but in a symbiotic relationship with eukaryotes. It was long thought that the lactic acid bacteria (which includes the guard dogs) were the most primative of bacteria since they don't have the genes for making so many things - none of the amino acids or bases, none of the growth factors we call vitamins. But, perhaps, the lactics are actually more evolved than things like E.coli and B.subtilis - because they have found in their symbiotic relationships that they can trash those genes because the host will supply them. The more genes eliminated the more efficiently they can live (at the cost of becoming an obligate symbiont). Like you said, Dr Indigo, among those trashed genes might be the μ-RNA's leaving the prokaryotes without them. They could leave that sort of control to the host plant or animal.

THE CONVERSATION OVER LUNCH.
Amy:	Say, did any of you see the article on micro-RNA's in Science News (ref)? These are very small RNA's that people previously thought were junk. But maybe not. If indeed the μ-RNA's are truly control elements, there must be several species of them, and the code within any one species must be fairly well conserved - certainly not random and free to mutate at will without any consequences to the organism.
Scarlet:	Ahhh, before we jump too far ahead of ourselves I think that we need to put all this into context. Wouldn't you think μ-RNA's might find themselves in the general subject are of biological controls just like genes are controllers? In fact, aren't all of the controls of a cell encoded in the genes?
Amy:	Scarlet! How can you say that? I would have expected you of all people to know that not everything in a cell is designed by genes and their protein products. I am aghast! Take membranes for just a trivial example.
Tony:	Calm down, Amy. Let's not get sarcastic. We don't need a cat-fight here. Remember, we have a guest sitting here with us. Membranes are hardly trivial. And I might add cell walls also - both of bacteria and plants, to your list of cell parts that are not genetically programmed.
Amy:	You're right, Prof. Indigo. Sorry, Scarlet. I guess all of us have holes in our knowledge. Let me try to explain it to you by giving you a classic experiment that has been known for at least 40 years. If you grow some E.coli in 12% sucrose (that's an isotonic solution for E.coli), you can dissolve off their cell walls with lysozyme. Because of its being isotonic, the membranes don't burst, and the cells change from being little sausage shapes into spheres that are imaginatively called spheroplasts. These spheroplasts continue happily growing and dividing in the presence of the sucrose and lysozyme. But look what happens when you remove the lysozyme by some biochemical trick: the spheroplast population just continues growing as spheroplasts! Not as sausages. The spheroplasts simply cannot make new cell wall. Yes, they are chock full of the building blocks for making cell wall, but they just don't come together. It was soon found that if you were to stir in the merest of pinches of purified E.coli cell wall to the spheroplast culture, within seconds the whole culture goes frantically about making cell walls and in a minute or two there are no spheroplasts left - all are now sausages! It's almost like adding a seed crystal to a super-cooled solution of salt or sugar. It almost explosively forms crystalline precipitates. Just needed a little starter material - sort of like showing the cells how to construct cell walls from all the building blocks they have already to go. So you see, Scarlet, the E.coli cell doesn't have the genes for the general plan of its cell wall. And the same is the case for all cells and their membranes. These are cases where the "plan" is stored in the stuff itself. Membranes beget membranes; cell walls beget cell walls.
You:	Hey, that gives me an idea for a project! I am so excited. Wonder what would happen if I added cell walls isolated from Bacillus to E.coli spheroplasts. Are all the right building blocks there? Or maybe Bacillus is too unrelated to E.coli. Perhaps I would be limited to some Gram-negative cell walls for "seed crystals."
Tony:	See, what did I tell you. We have great students! But I think we have strayed a bit. Let's get back to DNA-based genetics. Why don't I start jotting down things we know about micro-RNA's. Maybe a picture will come into focus as to where we should head our research. Okay, I'm ready. Who's got something for my list?
Scarlet:	Well, Dr. Indigo, if they are truly DNA transcripts, we might that they have a conserved genetic code - any given type of μ-RNA would all have the same sequence. And therefore we should expect to find their genes scattered somewhere on the chromosomes of their eukaryotic hosts. But where?
	Yes--, and do these genes take up much space as if they were either numbered in the thousands of species, or highly redundant as are the tRNA genes in the nucleolar organizer region...
Amy:	Yes, about 10,000 copies of each "t-gene" more or less.
Scarlet:	But where are these μ-RNA genes hiding on the chromosome? Unlike in T4 phage where somewhere like 90% of the chromosome can be accounted for in RNA-DNA hybridization studies, eukaryotic DNA seems to have a very low percentage of DNA complementary to large molecules of RNA. Does this mean that most of eukaryotic DNA is non-coding, or that the RNA isolated for hybridization studies was only from one of many, many different portions of the cell or organism's cycle. So my question stands: where are these μ-RNA genes located on the chromosomes?
You:	Introns? My teacher was just talking about introns - whatever happens to the snipped-out introns?
Tony:	Hmmm, I think you might have something there. Maybe the origin of some μ-RNA's is that they are excised introns. I believe that there are several bits of reasoning in support of this candidacy. Hmmm. Apparently, a minimum of 50% of eukaryotic cistrons consists of introns. This would be a shocker of apparent inefficiency if all these stretches of DNA and RNA were only to be thrown away. A huge portion of transcript RNA is clipped out and seemingly thrown away and digested. I say 'seemingly' because that discarding is not proven - just assumed. Let me scribble this down - good stuff!
Amy:	I was just reading somewhere something supporting the notion that these excised introns may serve a useful purpose. It was shown by a wonderful experiment. Two strains of SV40 (simian virus 40) were genetically engineered to contain the coding sequence for some human protein. In one strain, the inserted piece was normal human DNA with all the introns in it, and the other strain contained an inserted piece with all the introns excised out. These two strains were then used to infect a human cell culture. SV40 goes through two stages of infection/unwrapping. In the first it passes into the cytoplasm, and in the second it passes into the nucleus. Only the first strain made the human protein. Of course, an alternative value of the intron excision may only have to do with the linear transport of the transcripts out of the nuclear membrane and into the cytoplasm.
Scarlet:	Black, I might add another interesting factlet: the excision of introns is far from a random event: first one is excised and the site ligated, and then the next one is excised and ligated, and so on. And it always proceeds in the same order for a given gene. This is highly reminiscent of the "clock" mechanism in T4-phage that governs the passage of time in the infection cycle separating the cycle into early, middle and late phases. T4 does this by placing a different gene for one of the components of transcriptase at the end of each of the phase's stretch of DNA. When transcriptase-1 reaches that point, the gene is expressed as a protein that replaces one portion of transcriptase-1 converting it to transcriptase-2, which can only begin reading an operator at the beginning of the next phase's portion of DNA. So the new transcriptase-2 starts reading there at operator-2 (but not back at operator-1). Eventually it reaches another gene, which expresses a factor that converts transcriptase-2 to t'ase-3. Thus this phage uses as its clockwork escapement the time it takes transcription of a long, poly-cistronic gene sequence to be read.
Tony:	That sounds good. Let me get those thoughts down here on the list. You are saying that with the sequencial exision and ligation of introns, it sure looks as if the first intron fragment may be needed to do the next intron's exision, and so on. Thus the intron fragments (similar in size to μ-RNA's) may have at least one control function. Of course, to begin the exision of the first intron would require a "helper-RNA" from elsewhere.
Amy:	And that would mean that the introns must be highly conserved sequences. Are they?
Scarlet:	They are indeed! And they are indeed DNA transcripts.
Tony:	Slow down: I am having trouble getting all this down. I am really happy to have you young hotshots here - you can remember all the details. They escape an old guy like me.
Amy:	I would like to ask a series of questions dealing with the physiology of the transport of the transcripts through the nuclear membrane. As soon as one thinks of a long piece of linear RNA passing through a pore, one has a primative sort of production line - at least one station on a production line. As the linear piece continues to pass by, the pore enzymes do their snips and weldings - or ligations. But first the RNA must first be "capped" (as a needle on thread) just to be able to begin passing through the pore. Then the production line does its work. But the speed of the "production" must somehow be regulated. Also what must be regulated and balanced by feedback from the outside cytoplasm must be which transcripts are preferred to be capped and threaded through the pores.
Tony:	No, we are not at a loss for possible locations of μ-RNA genes or of their functions. We've got more items on this list that I thought we would be able to think of. So now to the nitty-gritty: what sorts of experiments can we propose to see if any of our speculations are correct. If we can hone our speculations into experimental questions, we finally have hypotheses! Oh, another thought just came to me: why don't prokaryotes show evidence of having introns? Some evolutionary microbiologists are known to say that microbes started out inefficient, and rapidly streamlined their various machinations for simplicity and rapidity of function so that if the environment favored them, they would be johnny-on-the-spot and be able to grow their populations quickly. Most eukaryotes don't seem to place that much emphasis on rapidity of growth - there is simply no way that they can beat the microbes to the punch. But eukaryotes do things better by specializing for being able to eat only certain things, or for poisoning or eating competitors.
You:	And then come the guard dog bacteria I've been learning about, which do kill competitors - but in a symbiotic relationship with eukaryotes. It was long thought that the lactic acid bacteria (which includes the guard dogs) were the most primative of bacteria since they don't have the genes for making so many things - none of the amino acids or bases, none of the growth factors we call vitamins. But, perhaps, the lactics are actually more evolved than things like E.coli and B.subtilis - because they have found in their symbiotic relationships that they can trash those genes because the host will supply them. The more genes eliminated the more efficiently they can live (at the cost of becoming an obligate symbiont). Like you said, Dr Indigo, among those trashed genes might be the μ-RNA's leaving the prokaryotes without them. They could leave that sort of control to the host plant or animal.

The group decided their minds had run dry, and the informal meeting broke up and each party went their way. But each person found that thoughts brought up kept recurring to them. Their eyes and ears were open and atuned now to anything else read or discussed in their presence that might apply to this μ-RNA subject. Further informal meetings were had, and even visitors were asked to join them. Before long various collaborations began.

This is how science very frequently begins - informal discussions and wonderings. And for you, the reader of this, you have learned a few facts that are as yet disconnected. But perhaps you might want to carry these forward and solve the bigger picture. Good luck!

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