Snapshots of the architecture of life

Roland Beckmann, Professor of Biochemistry at LMU’s Gene Center Munich, investigates fundamental processes of life that occur in every cell. The structural biologist is a specialist in cryogenic electron microscopy, which allows the complex architecture of biomolecules to be made visible at the atomic level. He is particularly interested in the structure and workings of ribosomes, the protein factories of the cell. Together with Ed Hurt from Heidelberg University, Beckmann has summarized how ribosomes are assembled in the cells of higher organisms in two “snapshot” articles, which have appeared in the renowned journal, Cell. We spoke to Professor Beckmann about his research.

What occasioned the snapshots?

Roland Beckmann: We’ve reached a point where we – my team and Ed Hurt’s working together – have a very good overall understanding of the processes of ribosome biogenesis in eukaryotic cells – that is to say, the assembly of these complicated molecular machines. We’ve clarified many structures of intermediate stages and can now visualize the majority of this process, from the beginning in the nucleolus – a spherical area inside the cell nucleus – to the nucleus, to the transport from the nucleus into the cytoplasm. In view of this completeness, it made sense to publish these snapshots now and provide an overview.

Why are ribosomes important?

Ribosomes are one of the most important molecular machines we have. During the process known as translation, ribosomes link amino acids together according to the genetic information encoded in RNA to form proteins. And proteins of course are vital for pretty much all functions in the cell.

But the ribosome does much more besides: We like to compare it to a smartphone, with which you can make calls but also do a thousand other things. Ribosomes play an important role in quality control, for example, protecting the cell against defective and harmful translation products and keeping the amount of RNA molecules in equilibrium.

This quality control is our second major research field after ribosome biogenesis. We discovered, for instance, that ribosomes react sensitively to stress, whereupon they stall and bump into each other during the translation process. They have traffic accidents, so to speak. And the cell is able to recognize this and trigger protective mechanisms. These collisions have proven to be a general principle that holds from bacterial to human cells. Very broadly, we can say that the ribosome is used as a sort of integrator for perceiving environmental influences, which can then be read by the cell to trigger the necessary reactions.

The snapshots summarize the results of ten years of research into ribosome biogenesis. What were the important milestones on this journey?

The ten years refer to the collaboration between my research group and Ed Hurt’s; I myself have been researching this topic for a good deal longer. The ribosome consists of a large and a small subunit, and our first major milestone, I would say, was when we elucidated the structure of a precursor of the small subunit, which had only been known biochemically. This so-called processome is interesting because it is extremely large. Indeed, it is larger than the full-grown ribosome and has a shape that allows the small subunit to be folded into it.

Subsequently, we found more and more intermediates and discovered that the small subunit peels off the processome like an onion, with one component after another being shed. And we’re also confident that a certain state of the small subunit is necessary for the peeling-off signal to be given.

And the large subunit?

With the large subunit, it was a similar story. At first, it was not at all clear how it starts to assemble. We identified very early intermediates and showed that first an exoskeleton is formed, which is filled up rather like you would fill a bowl. That was entirely unexpected.

In addition, we discovered a puzzling phenomenon whereby a certain RNA is incorporated into the large subunit the wrong way round. This RNA forms a sort of arch, which is initially turned round 180 degrees. Later, the position is corrected by means of rather complicated machinery. This backward integration, it turns out, has been preserved from bacteria to humans and is designed to prevent incorrect folding in the course of assembly.

Other results have shown that there is clearly a general principle by which the RNA is taken out of play, as it were, through the binding of certain factors, so that it does not get in the way of other areas when they are being folded. The final conformation is only adopted with the progressive completion of the ribosome.

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What makes working with ribosomes so challenging?

First of all, the intermediate products that arise during assembly are highly complex. These assembly intermediates consist of large RNAs and hundreds of proteins. It’s thought that human cells need 300 or more factors to make these complexes. It’s not so easy here to isolate individual substages, nor can you manufacture hundreds of factors and reproduce the assembly in vitro – particularly since assembly begins at the instant in which the ribosomal RNA is transcribed.

Moreover, we can’t really get at the very early intermediate products. This is partly because they are so unstructured that it’s scarcely possible to meaningfully analyze them, but also because it’s so hard to isolate them. Many intermediates are in the nucleolus, and that is one of the classic organelles without a membrane. The nucleolus represents a condensate with unique properties of its own, and it’s difficult to get at the corresponding molecules to study them.

On the other hand, ribosomes are otherwise very good objects for analysis using cryogenic electron microscopy, where the motto is: the bigger, the better.

Has technical progress played an important role over the past decade?

Absolutely. It was important for us to get a handle on the model systems by developing certain markers. Because we cannot assemble the intermediate stages, we have to isolate them using specific affinity tags. The problem is that the tags can be bound for rather a long time during the manufacture of the ribosome and migrate from the nucleolus to the cytoplasm along with various intermediates. As a result, you get a menagerie of intermediate products during the cleaning, which cannot be adequately sorted.

Ed Hurt invented beautiful systems that permit the use of two different markers. This is referred to as a split tag. It allows us, for example, to first tag one factor, which is bound to the intermediates from the nucleolus all the way into the nucleus, and then tag a second factor, which might be bound only from the end phase in the nucleus to the cytoplasm. In this way, we can isolate smaller subgroups. That was absolutely key to all the things we’ve done.

A second important aspect is that we started at a relatively early stage to work not only with yeast as a model system, but also with a thermophilic fungus. These fungi live in dung heaps, where temperatures can reach up to 50-60 degrees. Consequently, their proteins are adapted to heat and are much more stable and better to handle at room temperature than their yeast counterparts.

Have there also been advances in microscopy?

Yes, that was the second thing that was absolutely decisive. There have been huge technical advances in cryogenic electron microscopy. The first detectors had scintillators that generated a light pulse with each electron, which was then seen by the detector. These detectors were replaced by direct electron detectors, which no longer need a scintillator and can actually perceive each individual electron with pixel resolution.

This improved the quality to such a degree that molecular resolution – that is to say, three Ångström or better – is routinely achieved, which was previously unobtainable. The best resolutions approach one Ångström. And naturally this makes a massive difference. In addition, there have been new developments in software for the reconstruction and classification of particles.

Can defective ribosomes trigger diseases?

In principle, yes. Ribosomopathies do in fact exist. These diseases are based in most instances on mutations in ribosomal proteins or in assembly factors, which cause ribosomes to be formed more slowly or incorrectly. Astonishingly, most of these diseases affect blood formation, leading to anemias or leukemias.

Can your research help pave the way for new therapeutic approaches?

One of the reasons we started investigating this subject was the connection between ribosomopathies and defective assembly reactions. If we understand the mechanisms, we surmised, then it might lead to something. However, this is a difficult step to accomplish, because, as it transpired, a pathological phenotype is caused by the ribosome quantity being wrong. Correcting a deficit is rather tricky in this complex context. But in principle, interventions are certainly possible, if you have understood the mechanisms.

The faulty ribosomes are dismantled, leaving the cells without enough ribosomes. This presumably means that certain RNAs that do not occur frequently in the cell are no longer converted into proteins. Consequently, a different set of proteins is made than would be the case with a normal quantity of ribosomes. There are very many different mutations we are now aware of. This leads to an imbalance in translation, which we don’t yet understand well.

This is one question we’re studying. We want to investigate, for example, where these sick ribosomes end up. Are they all dismantled, or do some of them wind up in the translation cycle after all, where they might cause stress?

What questions remain to be resolved?

There are many open questions relating to ribosome biogenesis. A major problem is that although our structural investigations produce beautiful galleries, they are rather static and often we don’t know exactly how one state leads to another. Which signals are needed for the process to advance? This is quite difficult to address, partly because in-vitro investigations are so rarely successful. In fact, you can only really observe these things in the cell. In bacteria, we can reconstitute the process isolated in the test tube to a certain degree, but we haven’t accomplished this in eukaryotic cells yet. It would be interesting to really understand the transition that takes place in ribosome biogenesis and its incorporation into all possible signaling pathways.

And when it comes to translation, there’s also a lot we don’t yet understand. For example, how does the recognition process actually work when ribosomes collide. How do they realize that they have bumped into each other? How are the ensuing signaling pathways generated at the molecular level? And how are these signals related to each other? As you can see, there are many outstanding questions. Our work is far from done.

More on Roland Beckmann´s research

Ribosome assembly: The final trimming step

Ribosomes: Molecular wedge assists recycling

A molecular hammock for cotranslational modification