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Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively. Cells rely on a number of RNA degradation pathways to ensure correct and timely processing and turnover of both coding and non-coding RNAs. Another important function of RNA degradation is the rapid elimination of misprocessed RNA species, maturation by-products, and nonfunctional RNAs that are frequently produced by pervasive transcription.

In yeast and human, processing, turnover and surveillance of all nuclear exosome targets depend on a single RNA helicase, MTR4. MTR4 and HEN2 reside in nucleolar and nucleoplasmic compartments, respectively, and target different subsets of nuclear RNA substrates for degradation by the exosome.

The presence of both MTR4 and HEN2 homologues in green algae, mosses and land plants suggest that the functional duality of exosome-associated RNA helicases is evolutionarily conserved in the entire green lineage. The emerging picture is that, despite a high degree of sequence conservation, intracellular distribution, activities and functions of exosome cofactors vary considerably among different eukaryotes.

PLoS Genet 10 8 : e This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Efficient processing and degradation of RNA is a key process for the post-transcriptional control of gene expression. In vivo, exosome activity requires the interaction of the exosome complex with associated RNA helicases. In both yeast and human MTR4 is an essential protein required for all functions of the nuclear exosome [9] , [10].

We have previously shown that AtMTR4 encoded by At1g is a predominantly nucleolar protein required for the efficient degradation of misprocessed 5. The requirement for AtMTR4 in efficient rRNA production is reflected by the phenotype of mtr4 mutants, which show a characteristic combination of developmental growth defects also observed in ribosomal protein mutants and in other Arabidopsis mutants lacking putative ribosome biogenesis factors such as nucleolin [14] — [19].

HEN2 HUA enhancer 2 , At2g was originally identified in a genetic screen for mutations that enhance the flower morphology defects observed in hua1 and hua2 mutants [12]. A follow-up study showed that hen2 single mutants accumulate, as compared to wild type plants, slightly higher levels of a polyadenylated transcript comprising the two first exons and a large portion of the second intron of the AGAMOUS gene product, suggesting that the HEN2 protein could be involved in the degradation of misprocessed AGAMOUS transcripts [20].

We show here that HEN2 is a nucleoplasmic protein that is associated with the Arabidopsis exosome core complex and has a specific role in the exosome-mediated degradation of non-coding RNAs, misprocessed mRNAs, introns and transcripts derived from retrotransposons and non-genic regions. MTR4, by contrast, is associated with a distinct set of proteins, many of which appear to be involved in ribosome biogenesis.

Our results indicate a high degree of spatial and functional specialization of exosome activating RNA helicases in Arabidopsis. However, none of them has yet been shown to physically interact with the exosome. To better define the composition of plant exosome complexes, we used myc-tagged and GFP-tagged versions of the exosome core subunit RRP41 as baits in co-immunoprecipitation IP experiments.

RRP41 fusion proteins were expressed under the control of the 1 kb genomic region upstream of the endogenous RRP41 gene. To test this hypothesis and identify potential exosome co-factors, tagged-RRP41 and associated proteins were affinity-purified using superparamagnetic particles coated with anti-myc or anti-GFP monoclonal antibodies, respectively. A final list of 14 proteins was established by excluding proteins present in mock purifications and by crossing the datasets of three biological repeats Table 1.

In addition to the nine exosome subunits that were previously characterized [5] , five novel proteins were detected, albeit with lower number of spectra reflecting a lower abundance as compared to the canonical EXO9 subunits Table 1 , Table S1. A phylogenetic analysis of related proteins from animals, fungi and other eukaryotic clades revealed that most organisms possess both a single MTR4 and a single SKI2 protein; however, HEN2 homologues are restricted to the green lineage Fig. Protein sequences were retrieved from metazome, phytozome and JGI databases and aligned with ClustalX.

Dark red, vertebrates; light red, other eumetazoa; pink, M. S5 [13]. S5 , Fig. Distribution of GFP-fusion proteins in root cells of stable Arabidopsis transformants. Further data mining revealed that additional 12 proteins have a proven or predicted role in ribosome biogenesis Table 2. These data suggest that the functional link between exosome, CBC and NEXT complexes that was recently established in human cells [21] may be conserved in plants.

So far, our data suggested that HEN2 might operate as cofactor of the nucleoplasmic exosome complex. In order to investigate the function of HEN2 for the degradation of exosome substrates, we tested the accumulation of a pseudogene and five non-coding RNAs selected from the list of known polyadenylated plant exosome substrates [5]. Steady-state levels of the six selected exosome targets were determined by quantitative RT-PCR using oligo-dT primed cDNA samples prepared from seedlings of wild type, mtr4 - 1 , mtr4 - 2 , hen or hen mutant plants.

As shown in Fig. By contrast, no or only a mild accumulation was observed in mtr4 mutants. These data provided a first indication that HEN2 is involved in the degradation of nuclear exosome targets that are not substrates of AtMTR4. Steady-state levels of exosome targets selected from [5] , see also Fig. The histogram shows the fold change relative to wild type.

To evaluate the respective contribution of HEN2 and AtMTR4 to the degradation of nuclear exosome substrates in an unbiased manner, we determined the accumulation of polyadenylated transcripts using full-genome tiling microarray arrays. For this experiment, cDNA was prepared from two biological replicates of wild type, mtr and hen mutants. Each mutant sample was co-hybridized against a wild type sample to NimbleGen A. The microarray chip contains 1,, strand-specific probes covering both coding and non-coding regions with an average resolution of nt.

For each biological replicate, expression of each mutant was compared to the expression of the wild type. The statistical analysis, based on a 4-state Hidden Markov Chain, classified probes into four clusters corresponding to over-expressed probes, under-expressed probes, probes with unchanged expression, and noise not expressed probes , respectively. Interestingly, the analysis did not declare any probe as under-expressed in both biological replicates. Indeed, signals for unique probes were significantly increased in both biological replicates of hen2 samples.

A file allowing the visualization of the upregulated probes aligned to the Arabidopsis genome can be found in dataset S1. For the further analysis, we sorted the probes according to their genome coordinates to identify upregulated regions. Only regions with at least two consecutive probes were considered for interpretation. However, for the majority of the cases, regions, of which were only observed in hen2 samples, Table S4 , the upregulated transcripts were apparently not mature mRNAs. In fact, most of the upregulated regions corresponded to short portions of protein coding genes Table S4.

Furthermore, many upregulated regions contained both exonic and intronic sequences, suggesting the accumulation of incompletely spliced transcripts. To test this possibility, we compared by qRT-PCR the steady-state levels of individual exons, introns, regions comprising unspliced intron-exon junctions and correctly spliced transcripts from selected loci Fig.

For most loci, we detected the upregulation of both unspliced and spliced transcripts albeit to different levels, please note the scales in Fig. By contrast, a smaller product of only about bp was amplified from hen2 or RRP41 RNAi plants, and corresponded to a population of transcripts that comprised the unspliced donor site of the first intron Fig.

Of the 20 clones that were obtained from the hen sample 18 were polyadenylated at or close to the intron acceptor site Fig. S10 , indicating that they are indeed marked for degradation by the nuclear exosome. The remaining 2 clones had polyadenylation sites 8 and 52 nt upstream of the acceptor site and likely represent degradation intermediates Fig. A diagram of the genomic locus indicated by the respective AGI number is shown at the top of each panel. Red bars above the diagram represent probes detected in the tiling analysis. Please see legend of Fig.

The diagram shows the At1g locus. Red bars above the diagram represent probes detected in the microarray analysis. Please note the scales. A negative stain of PCR products separated on a 1. The upper band marked by a star corresponded to the fully spliced mRNA as depicted by the long orange arrow below the diagram. The tiling data suggested that HEN2 also participates in the elimination of excised introns.

In fact, 34 of the protein coding regions that were detected in the microarray analysis corresponded exclusively to intron regions Table S5 , 28 of which were only observed upon loss of HEN2. The upregulation of the pseudogene At1g Fig. Finally, some mRNAs were only detected in the very same samples that have been used for the microarray, but not in other mutant plants grown in the same culture conditions Fig.

This inconsistence was in sharp contrast to all other types of substrates that were tested in the course of the study, which were reproducibly detected in all replicates, in independent hen2 T-DNA insertion mutants, and in RRP41 RNAi lines. Therefore, we doubt that all of the mRNAs that were detected in our tiling analysis represent true substrates of exosome-mediated decay. Although nuclear degradation can probably contribute to mRNA degradation [3] , [5] , [48] , the upregulation of mRNAs can also be explained by indirect effects, e. Indeed, data mining revealed that many of the mRNAs detected by the tiling arrays are linked to stress response Table S Hence, the majority of the mRNAs that we detected by the tiling array are probably not bona fide substrates of HEN2 or the nuclear exosome.

By contrast, the upregulation of short mRNA-derived transcripts Fig. S9 , Fig. S10 , introns Fig. Please refer to the legend of Fig. The polyadenylated status of the accumulated transcripts indicates that they are tagged for degradation by the exosome, and indeed, all these classes of mRNA-derived transcripts have been described as exosome targets [5].

Hence, the most straightforward explanation for the accumulation of these transcripts in hen2 mutants is that HEN2 is required for the exosome-mediated elimination of different types of probably unfunctional RNAs that are generated from protein coding genes. Only a small number of such transcripts were observed in mtr4 mutants Table S4 , S5 and accumulated at lower levels Fig. Of the upregulated regions detected in the microarray analysis, regions mapped to non-coding regions. In fact, almost all of the snoRNA regions detected in our tiling array have also been previously identified as exosome substrates see Table S8 last column, and [5].

A diagram of the genomic locus with the indicated snoRNA genes is shown at the top of each panel. Individual snoRNA genes are represented as yellow arrows. Other non-coding RNA regions were also unequally distributed between hen2 and mtr4 samples. Similarly, we detected 29 putative antisense transcripts in hen2 samples, while only 2 potential antisense regions were upregulated in mtr4 samples Table S Cloning and sequencing of the PCR products revealed that 0 of 32 clones obtained from WT samples corresponded to the target sequence Fig.

By contrast, 28 of 32 clones obtained from hen2 samples and 3 of 32 clones obtained from mtr4 samples corresponded indeed to antisense transcripts derived from the target region Fig. Antisense sequences were polyadenylated, a hallmark of exosome-mediated RNA degradation, and between 67 and nt long Fig. These data strongly suggest that the antisense transcripts derived from the At5g locus are indeed substrates of polyadenylation-mediated decay facilitated by HEN2 and the RNA exosome.

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Finally, the microarray analysis detected 43 regions without any annotated genome features, including the intergenic repeat region on chromosome five that was already detected by our initial qRT-PCR experiments Fig. Similar to the distribution of non-coding RNA regions and potential antisense transcripts, the majority of the non-annotated regions 38 of 42 were exclusively observed in hen2 samples, while only 4 of 42 regions were found in mtr4 samples. A diagram of the At5g locus is shown at the top. A blue arrow represents the At5g mRNA.

Each of the orange horizontal bars below the diagram represents a polyadenylated clone obtained from the indicated sample. Taken together, the microarray analysis revealed that a large number of exosome targets, including short or incompletely spliced transcripts derived from mRNA genes, precursors and processing by-products of non-coding RNAs, and spurious transcripts generated from antisense and intergenic regions accumulate specifically in hen2 mutants Fig.

This indicates that HEN2 has a major function in the elimination of many different types of nuclear exosome substrates. A much smaller number of such non-ribosomal exosome substrates accumulated in mtr4 plants Fig. The transcriptomes of WT, hen and mtr plants were compared by whole genome microarrays. The histogram shows the total number of regions that were, as compared to wild type, overaccumulated in hen2 or mtr4 samples, respectively.

These data indicate that compromising exosome activity in nucleoli has only a limited effect on transgene S-PTGS. Mukherjee, D. Liu, H. Dodson, R. Regulation of pathways of mRNA destabilization and stabilization. Nucleic Acid Res. Moore, M. Nuclear RNA turnover. Muhlrad, D. Premature translational termination triggers mRNA decapping. Nature , — Cao, D.

Computational modeling and experimental analysis of nonsense-mediated decay in yeast. Cell 11 , — Takahashi, S. Frischmeyer, P. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons.

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Science , — Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. The transcription factor associated proteins Ccr4 and Caf1 are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Denis, C. Dlakic, M. Trends Biochem. Chen, J. Baggs, J. Nocturnin, a deadenylase in Xenopus laevis retina. A mechanism for posttranscriptional control of circadian-related mRNA. Daugeron, M. Nucleic Acids Res. Thore, S. X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex.

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EMBO Rep. Moser, M. Joyce, C. Polymerase structures and function: variations on a theme? Brown, C. Poly A tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Hammet, A. Ito, T. A comprehensive two-hybrid analysis to explore the yeast protein interactome. USA 98 , — Ren, Y. Lai, W. Tristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly A ribonuclease.

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Gao, M. Cell 5 , — The deadenylating nuclease DAN is involved in poly A tail removal during the meiotic maturation of Xenopus oocytes. Lejeune, F. Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Cell 12 , — Viswanathan, P. Dehlin, E. Cap-dependent deadenylation of mRNA. The mRNA cap structure stimulates rate of poly A removal and amplifies processivity of degradation. Sach, A. Translation initiation requires the PAB-dependent poly A ribonuclease in yeast. Cell 70 , — Wickens, M. Trends Genet.

Musing on the structural organization of the exosome complex. Butler, J. The yin and yang of the exosome. Trends Cell Biol. Symmons, M. A duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity, and regulation. Structure Fold Des.

Aloy, P. A complex prediction: three-dimensional model of the yeast exosome. Cell 91 , — Brown, J. With this official permission in my hand and ignoring the warnings of many friends that martial law was still in force, I flew from New York back to Warsaw in March , to take part in the Ph. By that time, I had already shipped reagents to Basel and hired two people for the new laboratory.

My family was due to move directly from New York into a rented apartment in Basel. The appointment was like a fairy tale, but reality and its complications soon caught up with us. Once in Warsaw, I was repeatedly denied a passport to travel to Basel, in fact to any symposium or seminar abroad. I suppose I should have expected the problem as I was twice prevented from traveling abroad for different periods during my student and Ph.

The situation looked hopeless, and hence Ola and the children arrived back in Warsaw in June. Over the next year, many people in Poland and abroad tried to help, but to no avail, and it became more worrying when I was even refused permission to travel to Moscow. Although this is hard to believe, the Moscow incident turned out to be the key to resolving my year-and-a-half detention in Poland. As I still hadn't received my passport by the day of the virology symposium, I cabled him that the Polish authorities were denying me permission to participate in his congress.

The reaction was instantaneous! The next morning, I received a call from the President of the Polish Academy of Sciences that my passport to travel to Moscow was ready. However, as the symposium had taken place the day before, I asked whether I could use the passport to travel to a meeting in The Netherlands starting a few days later, at which I was an invited speaker.

Surprisingly, they agreed, and I collected my passport from the Academy office. I noticed, however, that the passport had been re-stamped from multiple to single-exit status, and it was now obvious that the trip to The Netherlands would not be the end of my battle against travel restrictions. By now, we were quite paranoid: Ola and I met in the middle of a nearby sports field to discuss clandestinely how to proceed.

RNase MRP and the RNA processing cascade in the eukaryotic ancestor

At the invitation of our Spanish friends, Ola and the children had recently been granted a permit to travel to Spain for a vacation. Needless to say, my application for this trip had been rejected. Because the passport offices handling private and official trips were separate and possibly not in communication with each other, we decided to gamble and try to depart Poland on the same day, with me flying to Amsterdam and Ola taking the kids to Madrid.

The only obstacle to this plan was the urgent need for Spanish visas for my family. Alas, the Spanish Embassy in Warsaw was besieged by people who had been waiting for a visa for weeks. This is when my former association with the Nobel Prize recipient Severo Ochoa, a prominent figure in Spain, and my acting experience from my student days came in very handy. Thus, without telling anyone, not even our parents, we checked in separately a few days later and boarded our planes. At the meeting in Renesse, a Dutch resort on the North Sea, I offered a glass of beer to everybody in the audience of my after-dinner talk to celebrate the success of our conspiracy.

I then flew to Madrid, and a month later, we received our Swiss papers. The restrictions, this time for travel to Poland, remained in force for the next seven years. I felt very bad about abandoning my students in Warsaw, but my worries dissipated somewhat when Magda Konarska departed for a postdoc at MIT with Phil Sharp, the start of her illustrious career, and when Kazio Tyc Tycowski, another brilliant Warsaw student, left shortly afterward to join Joan Steitz's lab at Yale.

Forgive me this rather lengthy account of personal ups and downs in my early career. It makes perhaps more interesting reading than an everyday description of normal science which will follow. More important, however, the enforced improvisation in the lab, the solidarity of my friends and collaborators, and the decision to move to Basel all had profound effects on my science for the next 30 years. Luckily, the position as a research group leader and the attendant lab space were still waiting for me at the FMI in Basel.

Family-wise, events of the passing year were most difficult for our children. Moving between four countries, as well as changing languages and schools or kindergartens regularly, made them a bit confused. In Finland? After a few months of tutoring, our children, unlike their parents, were fluent in both German and the Swiss dialect.

As for my daughter Magdalena, I will talk about some of her activities further below. Despite my previous experience with plant viruses and enzymology, the world of plant physiology was totally alien to me, but I was fascinated by the richness of plant RNA-related phenomena, as exemplified by viroid RNAs and the RNA viruses with bizarre gene expression properties.

In addition, the genomes of plant mitochondria are much larger than those of vertebrates, and the processing of their transcripts is very complex. We were puzzled by the inability of plant cells to process mammalian pre-mRNAs despite the fact that plant and animal splice site consensus sequences are quite similar Greg Goodall, an excellent postdoc from Australia, approached the problem by assembling a large collection of synthetic mosaic genes and testing the requirements for their splicing in plant protoplasts.

It turned out that plant introns must be AU-rich throughout their length to be effectively excised. We found that introns in some other organisms are also much more AU-rich than flanking exons reviewed in Ref. We characterized several families of nuclear RNA-binding proteins that were probably responsible for the recognition of AU- or U-rich nucleotide stretches in plant introns and continued to study their activities until the late s Ref. In parallel, we investigated U-snRNA genes and their products to better understand the intricacies of plant splicing, focusing mainly on the model plant Arabidopsis thaliana.

Actually, the highlights of this work related not so much to the role of U-snRNAs in splicing but to the transcription of their genes. Franz demonstrated that it is possible to change the polymerase specificity of the promoter by manipulating the spacing between the two elements Even more significant, Tamas found that such a change had occurred during evolution. Tamas was able to convert the plant U3 gene into a functional pol II-transcribed gene by extending the promoter element spacing by one helical turn This was the first example of a gene that is transcribed by different polymerases in different organisms.

We identified two conserved His-containing catalytic motifs in the enzymes 45 , 46 , and they became the founding members of a large family of 2 H phosphodiesterases We concentrated more on the cyclase, with the ultimate aim of understanding its biological function. The cyclase work took many interesting turns, and we were side-tracked on many occasions. Purification of the human cyclase 50 enabled us to clone its cDNA 51 , and this, in turn, revealed that the genes encoding cyclases are highly conserved in all three kingdoms.

Pascal Genschik, a very gifted French postdoc, showed cyclases to be a novel protein family, although later crystallization of the enzyme revealed that subdomains of cyclase share an ancient fold with other proteins Identification of the cyclase gene in E. Regrettably, the gene which we named rtcA was not essential for growth The ORF immediately upstream of rtcA named rtcB encoded a protein that is also highly conserved evolutionarily.

I enjoyed this transitory return to bacterial genetics very much, bringing as it did fond memories of my Ph. However, the research on the rtcAB operon was also associated with probably the largest blunder of my career! When defining RtcA as a cyclase, it occurred to us that its co-regulated neighbor RtcB, despite lacking sequence signatures of any known enzymes, might be a cyclic phosphate-dependent RNA ligase. Clearly, we abandoned research on cyclase 20 years too soon! I will return for a moment to the time we were chasing the cyclase function and describe how this chase diverted us to the field of ribosome biogenesis.

When inspecting different genomes in the mids for sequences with similarity to the human and E. Proteins of one class, encompassing human and E. We were very excited to find that the Rcl1p protein was essential for the growth of S. An answer to the riddle came from inspection of the sequences of the Rcl1 family proteins: these lacked a conserved histidine equivalent to the E.

Although disappointed, we were still curious as to why the RCL1 gene is required for the growth of S. A two-hybrid screen identified a further novel protein Bms1p involved in pre-rRNA processing as an Rcl1p partner Depletion of Bms1p, the first GTPase found to function in pre-rRNA processing, resulted in a phenotype identical to that seen upon depletion of Rcl1p 64 — I was fortunate to have Tamas Kiss as a postdoc in the laboratory.

Tamas was a real maverick of RNA analysis. He identified several new intron-encoded U-snoRNAs with rather unusual properties e. It was shown later that some intronic U-snoRNAs can use alternative, splicing-independent processing pathways Ref. Tamas Kiss also studied RNase MRP mitochondrial RNA processing , demonstrating that it acts in both plants and mammals in ribosome biogenesis in the nucleolus rather than in the processing of primers for DNA replication in mitochondria 72 , 73 , as originally proposed by others.

It is quite evident above that the topics covered in the lab were quite eclectic. But there was always some logic behind the moves from one subject to another. I always thought it good and healthy to follow potentially interesting leads arising from current experiments and to change the topic from time to time, to satisfy our evolving curiosity and to learn new things.

Sticking to a problem for decades to understand its every detail was certainly not my favored philosophy. I believe that group members also enjoyed the rather broad coverage of RNA biology, particularly when working in an institute like the FMI that provided an excellent infrastructure and nurtured a great intellectual environment with lots of opportunities for collaboration.

In the late s, I decided we needed a more radical change in our research, for three reasons. First, the growing complexity of ribosome biogenesis, with hundreds of protein- and snoRNP-transacting factors, made me skeptical that we would soon, if ever, understand the molecular details of the process. Second, the absence of suitable in vitro systems made studies of plant transcription and mRNA splicing increasingly difficult.

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Third, we were rather tired of chasing the function of cyclases and cyclic phosphodiesterases. However, I changed my mind entirely in , when David Baulcombe's lab presented the first evidence of the formation of small interfering si RNAs in virus-infected plants The decision in the group to throw most of our resources into investigating RNAi and, somewhat later, miRNAs in mammalian cells heralded probably the most rewarding episode in my career. As we have summarized this work in a number of recent reviews 80 — 83 , I will discuss only very few aspects here.

With our experience in RNA-processing factors, we thought we were well-placed to enter the race. In fact, we lost that contest and published our first Dicer paper 84 several months after the ground-breaking work by Hannon's group 85 , but we were in a good position to study the Dicer mechanism. Haidi Zhang, a Ph. There were several models on offer, but none seemed satisfactory. Haidi again took the lead and tested, together with Fabrice Kolb and Lukasz Jaskiewicz, two dozen purified mutants of both Dicer and its bacterial prototype, the E.

It was gratifying that crystallographic studies of Dicer later fully confirmed the correctness of our model In addition to the mechanistic aspects, we also carried out several studies on the function of Dicer in mammalian cells reviewed in Ref. Domain organization of the human Dicer and a model of dsRNA processing by the enzyme. Arrows point to two independent catalytic sites in the enzyme processing center. Reprinted from Zhang, H.


Cell , 57—68 87 , with permission from Elsevier. The most intensive effort of the past 15 years or so in our lab has been on the mechanism of miRNA repression. This took off with the arrival in of Ramesh Pillai, a very imaginative and courageous postdoc who had just graduated from the University of Bern. Using both Argonaute protein tethering and miRNA reporter approaches, Ramesh obtained evidence that miRNAs not only induce mRNA deadenylation and decay as shown later but also inhibit protein synthesis at the level of translation 89 , We were fortunate to have excellent collaborators in our mechanistic studies, in particular Nahum Sonenberg and Elena Conti.

Our contacts and friendship with Nahum, who was also an alumnus of Shatkin's lab, dated back to the late s Fig. We were proud to have attracted this renowned expert in protein synthesis to the miRNA repression field, when the connection with translation had become obvious. Nahum's lab, with modest input from us, quickly developed in vitro systems recapitulating miRNA repression. These were invaluable for gaining biochemical insights into the miRNA mechanism, pointing to the effect on the initiation step of translation 92 , Together with Elena Conti, a wizard crystallographer, we embarked on a project aimed at a structural understanding of the repression.

From the work of several labs including those of Nahum and Elisa Izaurralde, as well as our own reviewed in Refs. Despite extensive progress, there is still much controversy about the exact mechanism of miRNA repression and the relative contributions of translational and mRNA decay effects 83 , Central CNOT1 region as a hub of protein-protein interactions important for miRNA repression and other types of post-transcriptional regulation.

Reprinted from Mathys, H. Cell 54, — 98 , with permission from Elsevier. For more details, see Refs.

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Other collaborative studies led us to projects of some medical relevance. One such project particularly dear to my heart involved my daughter Magdalena, at the time an M. I strongly recommend collaborating with your daughters: it is great fun Fig. My most important life and work companions: my son Peter, my wife Ola Wodnar-Filipowicz, and my daughter Magdalena Filipowicz Sinnreich. In another collaboration, I have very much enjoyed working at the FMI with Botond Roska, an excellent retinal physiologist and molecular biologist.

We first aimed at identifying light-regulated miRNAs in mouse retina and indeed found some, particularly among those expressed in photoreceptors But once again it was a side observation that turned out to be more exciting. Jacek Krol, a very gifted postdoc in the lab, found in collaboration with others that miRNAs in mouse retinal neurons turn over much faster than in other cell types and that the same applies to primary hippocampal and cortical neurons of the mouse brain. At that time, miRNAs were considered to be very stable molecules, but our observations indicated that this is not always the case.

Moreover, miRNA decay seemed to be regulated by neuronal activity In continued collaboration with Botond, we investigated the role of miRNAs in the function of adult retina. The results were quite stunning! Having had many problems myself with my own retinas, I was excited to learn the intricacies of retinal physiology and pathology. Despite being compelled, for age-related reasons, to gradually dismantle my laboratory, the last few years of my scientific activity have been full of very memorable moments.

In May , my official retirement from the FMI was marked with a small symposium that gathered together a dozen of my professional colleagues and friends as speakers, as well as most of my former and current students, postdocs, and research assistants. This was a very moving experience, although unfortunately saddened by the last-minute cancellation by Aaron Shatkin. In the fall of , with Aaron's health already deteriorating, most of the alumni of his lab met at the Rockefeller University in New York at a symposium organized by Hiro Furuichi and Jim Darnell.

Aaron passed away in June , and Nahum Sonenberg and I wrote a retrospective on his life for Science My farewell symposium at the FMI was also an appropriate moment to think about the past 30 years spent at the Institute. It was one of my strokes of luck that brought me to the place, with its remarkable staff and a multitude of superb students and postdocs selected from all over the world. I also admired and enjoyed the dynamism and the scientific diversity of the institute.

I started as part of the plant community but then switched to the epigenetics and RNA biology program, and also collaborated with neurobiologists and stem cell researchers. With great satisfaction, I experienced how the FMI is continuously evolving — through the hiring of new staff and the building of supportive infrastructure — and how, with a cutting-edge attitude, it meets new challenges in biomedical sciences. Combined with a three-month fellowship at Trinity College, the Cambridge sabbatical was a particularly unique experience, also bringing back memories of the ground-breaking discoveries made at the LMB.

Most important, such activities and various advisory functions continue to keep me almost as busy as before. My 50 years of research on RNA have allowed me to witness many exciting developments in the field. Following the discoveries of mosaic genes and splicing, RNA editing, and catalytic RNA, it seemed unlikely that RNA could surprise us with more secrets of comparable caliber. We were wrong! I was also totally wrong in suspecting that we will never understand the molecular details of spliceosome dynamics or ribosome biogenesis.

High resolution cryo-electron microscopy has changed it all! Doing RNA research in the last few decades has been very rewarding and full of fun.