Where RNA Goes to Die

The exosome, which exorcises aged, defective, and short-lived RNAs, and also plays a part in maturation of ribosomal RNAs.

Much of the excitement in biology focuses on biosynthesis- the duplication of DNA, the growth of cells and organisms. But intrinsic to a dynamic, complex system like our cells are mechanisms to get rid of trash, which we produce in abundance. Trashy chemicals (like drugs and other complex toxins) are broken down by special enzymes in the liver. Old and broken-down proteins are sent to a cellular structure called the proteosome. And RNA also has a life cycle, which ends up at a small structure called the exosome.

RNA is intrinsically an unstable molecule, less chemically stable than DNA, which as archeologists, even paleotologists, have been finding, can survive for millions of years. But on a cellular time span, the spontaneous decay of RNA is hardly fast enough to provide critical regulation over key messages, which may need to be turned down in a matter of minutes. On the other hand, sometimes RNAs start out badly, with errors that jam up the translational machinery- there are several mechanisms in cells to figure out which RNAs should have short half-lives, are damaged, and are causing havoc, which generally send them to the exosome for degradation.

Human cells are full of RNA, much of it junk. By far the vast majority of RNA in a cell (90%) is ribosomal RNA and tRNA- the structural and functional cores of protein translation. Since translation is a rather slow and inefficient process, (as inherited from some extremely early ancestor), cells need tons of ribosomes, which typically make up a fair fraction of the dry mass of cells- up to 30% in bacteria. Exosomes are key processing centers that trim the ribsomal RNA and degrade excess bits. But other classes of cellular RNA are more diverse and interesting. Indeed, the encode project and similar projects have found that most of our genomes are transcribed to RNA. Some of these newly found RNAs are functional, but most is junk- junk RNA transcribed by a smart, but rather promiscuous transcription process, into junk RNA. Which does little harm if it gets sent right into the trash can.

A recent paper extended a lengthy trail of work into the structure of the exosome, reconstituting the full complexes from human and from yeast, and obtaining a new structure from the human form. Reconstitution means that the complex was not purified from cells or tissues as a whole, but that the individual proteins composing it were purified, here from bacterial cells or human cells carrying the encoding genes, and then later mixed to produce the full complex of about nine proteins.

Straight from the bench. Proteins are stained/displayed in blue, sorted by their molecular size by being electrophoresed (driven by an electric voltage) through a gel, from top to bottom. Smaller proteins (markers on left, marked in kilo Daltons) head toward the bottom. The middle lane is the yeast reconstituted exosome, the right lane is the human complex. The ten proteins of each complex are marked on the far right.

The complexes were then put into an electron microscope to get detailed pictures, which through the magic of superposition, averaging, and atomic modeling, can infer atomic-scale structures. The also created inactive mutatants of key proteins in the complexes, and then could add RNA to them to visualize where the RNA lies in the complex.

They found, as you can see, that the human and yeast complexes are very similar. The paths that the RNA takes within them is similar, but a bit longer in the human form, due to some accessory proteins like hMMP6, which protects a bit more of the RNA threaded through the middle from outside access. The active ribonucleases in all this are hRRP6, positioned towards the top right of the shown complexes, in red, and hDIS3, at the bottom in pink. hDIS3 is a processive RNAse, working from the 3' end to the 5' end and is also an endonuclease, meaning that it can cut in the middle of an RNA if necessary, while hRRP6 is less processive, and can nibble when needed. Also in the complex is a helicase (hMTR4) that can unwind double-standed RNAs, which is a frequent natural condition. One can see that these activities make up a directional machine, with RNAs getting fed into the top and consumed at the bottom (and side, if there is a second strand). The helicase opens the possibility of extracting jammed RNAs from polymerases and ribosomes, as though they were office printers. Enhancer RNAs, a recently-recognized class of junky RNA made at gene regulatory elements accumulates in hybrids to the enhancer DNA if not rapidly pulled off and degraded by exosomes.

Exosome structures from yeast (right) and human (left and middle). RNA is in black on right, and in schematic turquoise on left. The main digesting nuclease is at bottom in pink, while the unwinding helicase is in blue at top.

As mentioned above, chopping up RNA is not very chemically difficult. The hard part is controlling this process to separate the sheep from the goats. There is lots of good, even essential, RNA that needs to be kept around in the cell for as long as possible. All that ribosomal and rRNA is expensive to produce and should not be recycled until absolutely necessary. What regulates access to the exosome?

• Normal RNA processing prevents degradation. For example, as protein-coding mRNAs are made, they are spliced (introns removed) and polyadenylated and 5' O-methyl capped. The latter two processes protect them from degradation. Indeed, the length of the poly-A tail is in rough terms a timer for the livespan of that RNA, with longer tails on longer-lived mRNAs. Any problem in transcription and subsequent processing delays the addition of these markers and opens that RNA to attack by proteins that find uncapped and under-adenlyated RNAs and ferry them to exosomes.
• Exosomes are part of normal RNA processing in some cases. Ribosomal RNAs are trimmed by exosome versions, up to a point, where they are released and then modified and further processed into the pre-ribosomal structures, all in the nucleosome. The same goes for other small stable RNAs. Their mature structures are evidently stable enough to prevent excessive degradation. Special protein factors may facilitate this regulation as well, preventing, instead of encouraging, exosome activity beyond a certain point.
• Other special features of RNAs may be recognized by helper proteins that bring such RNAs to the exosome for degradation. For example, a complex called "PPC" recognizes short poly-A sequences, as would be common for timed-out normal mRNAs and many other RNA polymerase II transcripts that are not fully processed or stabilized by other means. It plays a big role in degrading lots of the junky RNA made from miscelleneous regions of the genome.

The cell is a sustainable chemical system, and part of that means having ways to dispose of trash. The exosome is the primary RNA disposal unit in eukaryotic cells, and has enabled the rather promiscuous transcription that has misled some people to think that we have far more genes or functional genetic elements than are actually there.

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