It's A Small nuclear RNA World After All December 10, 1999

Scientists enjoy change. We have to. Science changes and what we thought we knew yesterday is overtaken by what we know today. And what we know tomorrow will change what we know today.

Now, most of these changes are evolutionary in nature. They bring into focus aspects of our knowledge that are fuzzy and ill-defined. However, once in a while, new scientific facts require the old picture to be tossed in the trashcan and a new picture to constructed. When this revolution occurs, it is hard to ignore it. Not because it is new or different or because it is controversial. Scientists stand up and take notice because it explains so much. Catalytic RNA has spawned such an upheaval in scientific thought.

The Central Dogma in molecular genetics was first postulated by Crick. It stated that "...genetic information flows from DNA to RNA to protein." (Luckily I never throw anything away and was able to get this quote from my copy of Lehninger, 2nd Ed., 1975). Crick was among the first to recognize that the duplex nature of DNA allowed mRNA to be transcribed and then to be translated into protein by 'adapter' molecules called tRNA. The Central Dogma has been modified in the intervening years, particularly with the discovery of reverse transcriptases that create DNA from RNA, but it continues to have a huge impact on our view of the cell. People still concentrate on the beginning and the end of the information flow, and ignore the middle part. I will discuss reasons why RNA is more than just a link between DNA and protein but may be 'central' to the origin of life.

This revolution can be tied to a single observation. Part of the fun of writing a column is the ego-boost that can come from name-dropping. And I will do some shortly. But my purpose is to play off of the title of this essay. Research science IS a small world and many of us only 1 or 2 steps removed from almost every important scientific advancement.

Just down the hallway from where I was doing my post-doc at the University of Colorado in Boulder was a lab that studied an obscure organism called Tetrahymena. A research associate named Art Zaug kept getting some sort of contaminant in his DNA preps. Thinking "That's funny", he brought it to the attention of the principal investigator by the name of Tom Cech. Thus were ribozymes discovered. Stable RNA molecules that have enzymatic activities. RNA was not some sort of passive carrier but could actually perform biologically relevant reactions.

In the intervening years there has been an explosion in our understanding of the importance of these RNA-driven interactions. RNA can carry information and perform a wide-range of enzymatic activities. It can do this in the absence of DNA and protein. Theories now state that RNA was the original biomolecule in an RNA World. DNA and protein came later. This hypothesis answers many of the questions about early life and explains several riddles we have seen in modern biology.

Are there any RNAs in today's world that might give us some hint into the RNA World that existed in the early days of life on earth? Ribonucleoproteins (RNPs) containing both RNA and protein were known since the early '70s, but the emphasis of research was more on the protein than on the RNA. The RNA in a ribosome was to provide structure and a device to properly align mRNAs. Recent investigations indicate that this is a naive approach.

Eukaryotic ribosomes consist of 2 subunits, containing at least 3 rRNA molecules, ranging in size from 120 to 2900 nucleotides in length, and over 50 proteins. It is probably the most complex system routinely studied and is very ancient. Fossil cyanobacteria 3 billion years old appear to contain ribosomes. In the last few months, several papers have been published which delineate the structures of the small 30S subunit, the larger 50S and the complete 70S. Cryo-electron microscopy and X-ray diffraction techniques have been used to models at roughly 5 Å resolution. We can see where initiation factors bind, where elongation factors interact, where the nascent chain emerges from the ribosome. However, there is a major conundrum that still remains.

The ribosome has a single intrinsic biological activity, peptidyl-transferase. This is the actual joining of the nascent chain with an amino acid proffered by the tRNA. None of the ribosomal proteins has this activity, despite intense investigations. Although the recent structures do not clarify this, they do indicate that the most likely moiety involved in peptide bond formation is rRNA. In fact, the other 2 processes in the translational elongation cycle, aminoacyl-tRNA selection and translocation, are intrinsic to the ribosome, do not need extrinsic factors and may be facilitated solely by rRNA. This is topsy-turvy from the classic view. The rRNA is not a scaffold, it is the enzyme. It may very well be that rRNA is a relic of the RNA World. It originally developed in this ancient world and was co-opted into the modern world when DNA and protein appeared.

This idea that relics of the RNA World exist today was recently expanded in a paper in Bioessays. Are there any RNAs involved in metabolic processes? It turns out that there are several and almost all of them are involved with the same central complex, the ribosome. We have already discussed rRNA. RNase P is an enzyme that processes pre-tRNAs to yield mature molecules. Although there is protein present, it is the RNA moiety of RNase P that is the catalytic unit.

Another role for RNAs is in the splicing of mRNA. It has been known for some time that small nuclear RNAs (snRNAs) are important for proper splicing, and the subsequent removal of introns. The evidence for this predates ribozymes by quite a few years. Again, people have tended to concentrate on the proteins but the importance of the RNA is becoming more obvious. Because it is not only the snRNAs of the spliceosome that are important. Introns themselves are important.

The nucleolus is the organizing body where rRNA is created and modified in the eukaryotic cell. rRNA is highly processed before it becomes part of a ribosome. In eukaryotes, a long pre-rRNA molecule is cleaved into the 18S, 5.8S and 28S forms. Small nucleolar RNAs (snoRNAs) are responsible for this processing. snoRNAs are also responsible for the considerable modification rRNA undergoes (i.e. methylation and creation of pseudouridine). Surprisingly, many snoRNAs are created from processed introns. Often the 'host' gene for the snoRNAs is a ribosomal protein or translation factor. In one case, a single pre-mRNA has 8 different snoRNAs present in its introns and the spliced message is NEVER translated. The whole purpose of the exons is to provide the spliceosome ends to release the biologically important introns. So, splicing of exons using snRNAs is not only important for proper creation of mRNA necessary for the generation of protein by ribosomes, it actual creates important RNA molecules necessary for the proper processing and modification of rRNA. All these processes are linked and they all use RNA. This is just the sort of RNA-centric reactions one would expect in an RNA World or its relics.

In an ancient world, perhaps snRNAs released snoRNAs from template RNA, resulting in processing of rRNA that proceeded to create template RNA for snRNAs to process. No need for protein at all. The fact that even non-specific proteins help speed up ribozyme reactions suggests a plausible reason for proteins to evolve and eventually take on many of the RNA World functions.

An interesting consequence of this theory is that eukaryotic cells retain more of the characteristics of the "last universal common ancestor" (LUCA) than prokaryotic cells. Although this is counter-intuitive to how most of us think (i.e. prokaryotic came first followed by eukaryotic), it does simplify things. In an RNA World, RNA would be processed by RNA. In today's world, there is only one place where tRNA, rRNA and mRNA are all processed by complexes containing RNA.That is a eukaryotic cell. It is a lot easier to see how LUCA might lose such things as splicing and snoRNAs due to selective pressures than it is to imagine how splicing or rRNA processing evolved after catalytic proteins appeared. Proteins alone are much better at many things than ribozymes or RNA. Why would such a jury-rigged system evolve from a prokaryotic system that already functioned well? This has been a conundrum that has not easily been answered previously. However, viewing the processing of RNA as a relic of an ancient world provides a straight-forward hypothesis. How does it explain other differences between eukaryotic and prokaryotic cells?

Bacteria are selected for fast response time and growth. Replacement of RNA catalysis with proteins which are more efficient would have obvious selective advantages. RNA is very thermolabile but replacement of RNA processing by thermostable proteins would allow bacteria to exploit environments that eukaryotic cells could not. In addition, removing RNA processing, such as splicing, would also decrease the size of the genome, speeding up replication rates.

Most bacteria have circular genomes. Circular genomes have no ends to unravel. Most eukaryotic genomes are linear. To evolve from circular genomes to linear introduces a host of problems. The ends of eukaryotic chromosomes, the telomeres, are protected from degradation by an enzyme called telomerase. Is the presence of linear genomic material in eukaryotic cells another relic from LUCA? The telomerase is actually a ribonucleoprotein. It has an RNA component that is absolutely required for activity. In this case, it does not appear to have catalytic activity itself. The protein segment actually carries out the enzymatic reaction but guess what? This protein is a reverse transcriptase, capable of taking an RNA template and making DNA. The molecule that first violated the Central Dogma.

So, now we can see a way for DNA to enter the picture. Eukaryotic cells may have retained more of the characteristics of the first life on Earth than prokaryotic cells have. And who was the first to clone the catalytic subunit of the telomerase RNP and show it acted as a reverse transcriptase, bringing full circle the downfall of the Central Dogma? None other than Tom Cech's lab. It really is a small nuclear RNA World after all.

References

Anthony Poole, Daniel Jeffares, and David Penny. Early Evolution: Prokayotes, the new kids on the block. Bioessays 1999 21:880.

Joachim Lingner, Timothy R. Hughes, Andrej Shevchenko, Matthias Mann, Victoria Lundblad, Thomas R. Cech. Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase. Science 1997 276:561

Ante Tocilj, Frank Schlünzen, Daniela Janell, Marco Glühmann, Harly A. S. Hansen, Jörg Harms, Anat Bashan, Heike Bartels, Ilana Agmon, Francois Franceschi, and Ada Yonath, The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: Pattern fittings and the identification of a functional site. PNAS Dec 7 1999 96: 14252.

Jamie H. Cate, Marat M. Yusupov, Gulnara Zh. Yusupova, Thomas N. Earnest, and Harry F. Noller. X-ray Crystal Structures of 70S Ribosome Functional Complexes. Science 1999 285: 2095.

William M. Clemons Jr, Joanna L. C. May, Brian T. Wimberly, John P. Mccutcheon, Malcolm S. Capel and V. Ramakrishnan. Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution. Nature 1999 400: 833.

Nenad Ban, Poul Nissen, Jeffrey Hansen, Malcolm Capel, Peter B. Moore and Thomas A. Steitz. Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit. Nature 1999 400: 841.