Sixty years of life with François Gros.

This is meant to be a personal account of my relationship with François Gros who, as the director of the laboratory where I had my first team became a close and fatherly friend. Over 60 years we had permanent interactions, whether close or far away. And I wish to revive here some of our relation at the grassroots, as well as the folklore in and around his laboratory. He was not only an excellent scientist but also a Statesman of Science and beyond. Myself a minor figure in his big endeavors, this aspect of his life I just observed from below but with much empathy. All along he considered our experimental data and ideas realistic while they were rejected as iconoclastic by the many others. Being taken seriously as a researcher, I did not participate in his professional networks because I never fit the necessary profile in terms of scientific consensus. But his friendship was a major and basic element in my scientific and personal life; I hope my report hereafter does justice to his personality and immense merits.

Ceci est le souvenir très personnel de mes relations pendant 60 ans avec François Gros. Il m'avait accueilli dans son laboratoire ou j'avais ­ pour la première fois ­ une équipe de chercheurs autour de moi. Dans les 60 années suivantes, il m'est devenu un ami «  paternel  ¼ et nous avions des interactions de près ou de loin. Je me propose de décrire notre relation personnelle à la base et aussi le folklore dans et autour de son laboratoire. Il n'était pas seulement un chercheur de premier plan mais aussi un «  statesman of science  ¼, et un homme politique français et international. Moi, figure périphérique dans ses entreprises, je pouvais observer cet aspect de sa vie de façon passive mais avec beaucoup d'empathie. Ce qui était unique et déterminant pour moi et mes collaborateurs était le fait qu'il suivait avec intérêt et soutenait nos recherches considérées ­ à l'époque ­ comme sans base réaliste et même «  iconoclastes  ¼ par la majorité de nos collègues et compétiteurs. Malgré ma bonne réputation internationale comme chercheur, je ne pouvais pas faire partie de ses réseaux professionnels, car je n'opérais pas dans le cadre du consensus général dans les commissions scientifiques et de prix ­ garantes de reconnaissance «  officielle  ¼. Mais l'amitié de François était une part majeure et essentielle de ma vie scientifique et privée  ; et j'espère que mes souvenirs racontés ici feront justice à sa personnalité et ses mérites.

Is RNA the working genome in eukaryotes ? The 60 year evolution of a conceptual challenge.

About 80 years ago, in 1943, after a century of biochemical and genetic research, DNA was established as the carrier of genetic information. At the onset of Molecular Biology around 1960, the genome of living organisms embodied 3 basic, still unknown paradigms: its composition, organisation and expression. Between 1980 and 1990, its replication was understood, and ideas about its 3D-organisation were suggested and finally confirmed by 2010. The basic mechanisms of gene expression in higher organisms, the synthesis of precursor RNAs and their processing into functional RNAs, were also discovered about 60 years ago in 1961/62. However, some aspects were then, and are still now debated, although the latest results in post-genomic research have confirmed the basic principles. When my history-essay was published in 2003, describing the discovery of RNA processing 40 years earlier, the main facts were not yet generally confirmed or acknowledged. The processing of pre-rRNA to 28 S and 18 S rRNA was clearly demonstrated, confirmed by others and generally accepted as a fact. However, the "giant" size of pre-mRNA 10-100 kb-long and pervasive DNA transcription were still to be confirmed by post-genomic methods. It was found, surprisingly, that up to 90% of DNA is transcribed in the life cycle of eukaryotic organisms thus showing that pervasive transcription was the general rule. In this essay, we shall take a journey through the 60-year history of evolving paradigms of gene expression which followed the emergence of Molecular Biology, and we will also evoke some of the "folklore" in research throughout this period. Most important was the growing recognition that although the genome is encoded in DNA, the Working Genome in eukaryotic organisms is RNA.

Primary transcripts: From the discovery of RNA processing to current concepts of gene expression - Review.

The main purpose of this review is to recall for investigators - and in particular students -, some of the early data and concepts in molecular genetics and biology that are rarely cited in the current literature and are thus invariably overlooked. There is a growing tendency among editors and reviewers to consider that only data produced in the last 10-20 years or so are pertinent. However this is not the case. In exact science, sound data and lucid interpretation never become obsolete, and even if forgotten, will resurface sooner or later. In the field of gene expression, covered in the present review, recent post-genomic data have indeed confirmed many of the earlier results and concepts developed in the mid-seventies, well before the start of the recombinant DNA revolution. Human brains and even the most powerful computers, have difficulty in handling and making sense of the overwhelming flow of data generated by recent high-throughput technologies. This was easier when low throughput, more integrative methods based on biochemistry and microscopy dominated biological research. Nowadays, the need for organising concepts is ever more important, otherwise the mass of available data can generate only "building ruins" - the bricks without an architect. Concepts such as pervasive transcription of genomes, large genomic domains, full domain transcripts (FDTs) up to 100 kb long, the prevalence of post-transcriptional events in regulating eukaryotic gene expression, and the 3D-genome architecture, were all developed and discussed before 1990, and are only now coming back into vogue. Thus, to review the impact of earlier concepts on later developments in the field, I will confront former and current data and ideas, including a discussion of old and new methods. Whenever useful, I shall first briefly report post-genomic developments before addressing former results and interpretations. Equally important, some of the terms often used sloppily in scientific discussions will be clearly defined. As a basis for the ensuing discussion, some of the issues and facts related to eukaryotic gene expression will first be introduced. In chapter 2 the evolution in perception of biology over the last 60 years and the impact of the recombinant DNA revolution will be considered. Then, in chapter 3 data and theory concerning the genome, gene expression and genetics will be reviewed. The experimental and theoretical definition of the gene will be discussed before considering the 3 different types of genetic information - the "Triad" - and the importance of post-transcriptional regulation of gene expression in the light of the recent finding that 90% of genomic DNA seems to be transcribed. Some previous attempts to provide a conceptual framework for these observations will be recalled, in particular the "Cascade Regulation Hypothesis" (CRH) developed in 1967-85, and the "Gene and Genon" concept proposed in 2007. A knowledge of the size of primary transcripts is of prime importance, both for experimental and theoretical reasons, since these molecules represent the primary units of the "RNA genome" on which most of the post-transcriptional regulation of gene expression occurs. In chapter 4, I will first discuss some current post-genomic topics before summarising the discovery of the high Mr-RNA transcripts, and the investigation of their processing spanning the last 50 years. Since even today, a consensus concerning the real form of primary transcripts in eukaryotic cells has not yet been reached, I will refer to the viral and specialized cellular models which helped early on to understand the mechanisms of RNA processing and differential splicing which operate in cells and tissues. As a well-studied example of expression and regulation of a specific cellular gene in relation to differentiation and pathology, I will discuss the early and recent work on expression of the globin genes in nucleated avian erythroblasts. An important concept is that the primary transcript not only embodies protein-coding information and regulation of its expression, but also the 3D-structure of the genomic DNA from which it was derived. The wealth of recent post-genomic data published in this field emphasises the importance of a fundamental principle of genome organisation and expression that has been overlooked for years even though it was already discussed in the 1970-80ties. These issues are addressed in chapter 5 which focuses on the involvement of the nuclear matrix and nuclear architecture in DNA and RNA biology. This section will make reference to the Unified Matrix Hypothesis (UMH), which was the first molecular model of the 3D organisation of DNA and RNA. The chapter on the "RNA-genome and peripheral memories" discusses experimental data on the ribonucleoprotein complexes containing pre-mRNA (pre-mRNPs) and mRNA (mRNPs) which are organised in nuclear and cytoplasmic spaces respectively. Finally, "Outlook " will enumerate currently unresolved questions in the field, and will propose some ideas that may encourage further investigation, and comprehension of available experimental data still in need of interpretation. In chapter 8, some propositions and paradigms basic to the authors own analysis are discussed. "In conclusion" the raison d'être of this review is recalled and positioned within the overall framework of scientific endeavour.

Information theory, gene expression, and combinatorial regulation: a quantitative analysis.

According to a functional definition of the term "gene", a protein-coding gene corresponds to a polypeptide and, hence, a coding sequence. It is therefore as such not yet present at the DNA level, but assembled from possibly heterogeneous pieces in the course of RNA processing. Assembly and regulation of genes require, thus, information about when and in which quantity specific polypeptides are to be produced. To assess this, we draw upon precise biochemical data. On the basis of our conceptual framework, we also develop formal models for the coordinated expression of specific sets of genes through the interaction of transcripts and mRNAs and with proteins via a precise putative regulatory code. Thus, the nucleotides in transcripts and mRNA are not only arranged into amino acid-coding triplets, but at the same time may participate in regulatory oligomotifs that provide binding sites for specific proteins. We can then quantify and compare product and regulatory information involved in gene expression and regulation.

Regulation of gene expression and the transcription factor cycle hypothesis.

Post-genomic data show unexpected extent of the transcribed genome and the size of individual primary transcripts. Hence, most cis-regulatory modules (CRMs) binding transcription factors (TFs) at promotor, enhancer and other sites are actually transcribed within full domain transcripts (FDTs). The ensemble of these CRMs placed way upstream of exon clusters, downstream and in intronic or intergenic positions represent a program of gene expression which has been formally analysed within the Gene and Genon concept [1,2]. This concept has emphasised the necessity to separate product information from regulative information to allow information-theoretic analysis of gene expression. Classically, TFs have been assumed to act at DNA level exclusively but evidence has accumulated indicating eventual post-transcriptional functions. The transcription factor cycle (TFC) hypothesis suggests the transfer of DNA-bound factors to nascent RNA. Exerting downstream functions in RNA processing and transport, these factors would be liberated by RNA processing and cycle back to the DNA maintaining active transcription. Sequestered on RNA in absence of processing they would constitute a negative feedback loop. The TFC concept may explain epigenetic regulation in mitosis and meiosis. In mitosis control factors may survive as single proteins but also attached to FDTs as organised complexes. This process might perpetuate in cell division conditioning of chromatin for transcription. As observed on lampbrush chromosomes formed in avian and amphibian oogenesis, in meiosis the genome is fully transcribed and oocytes conserve high Mr RNA of high sequence complexity. When new interphase chromosomes form in daughter cells and early embryogenesis, TFs and other factors attached to RNA might be reinserted onto the DNA.

Gene and genon concept: coding versus regulation. A conceptual and information-theoretic analysis of genetic storage and expression in the light of modern molecular biology.

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term "genon". In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon.

The gene and the genon concept: a functional and information-theoretic analysis.

'Gene' has become a vague and ill-defined concept. To set the stage for mathematical analysis of gene storage and expression, we return to the original concept of the gene as a function encoded in the genome, basis of genetic analysis, that is a polypeptide or other functional product. The additional information needed to express a gene is contained within each mRNA as an ensemble of signals, added to or superimposed onto the coding sequence. To designate this programme, we introduce the term 'genon'. Individual genons are contained in the pre-mRNA forming a pre-genon. A genomic domain contains a proto-genon, with the signals of transcription activation in addition to the pre-genon in the transcripts. Some contain several mRNAs and hence genons, to be singled out by RNA processing and differential splicing. The programme in the genon in cis is implemented by corresponding factors of protein or RNA nature contained in the transgenon of the cell or organism. The gene, the cis programme contained in the individual domain and transcript, and the trans programme of factors, can be analysed by information theory.

RNA-dependent nuclear matrix contains a 33 kb globin full domain transcript as well as prosomes but no 26S proteasomes.

Previously, we have shown that in murine myoblasts prosomes are constituents of the nuclear matrix; a major part of the latter was found to be RNase sensitive. Here, we further define the RNA-dependent matrix in avian erythroblastosis virus (AEV) transformed erythroid cells in relation to its structure, presence of specific RNA, prosomes and/or proteasomes. These cells transcribe but do not express globin genes prior to induction. Electron micrographs show little difference in matrices treated with DNase alone or with both, DNase and RNase. In situ hybridization with alpha globin riboprobes shows that this matrix includes globin transcripts. Of particular interest is that, apparently, a nearly 35 kb long globin full domain transcript (FDT), including genes, intergenic regions and a large upstream domain is a part of the RNA-dependent nuclear matrix. The 23K-type of prosomes, previously shown to be co-localized with globin transcripts in the nuclear RNA processing centers, were found all over the nuclear matrix. Other types of prosomes show different distributions in the intact cell but similar distribution patterns on the matrix. Globin transcripts and at least 80% of prosomes disappear from matrices upon RNase treatment. Interestingly, the 19S proteasome modulator complex is insensitive to RNase treatment. Only 20S prosomes but not 26S proteasomes are thus part of the RNA-dependent nuclear matrix. We suggest that giant pre-mRNA and FDTs in processing, aligning prosomes and other RNA-binding proteins are involved in the organization of the dynamic nuclear matrix. It is proposed that the putative function of RNA within the nuclear matrix and, thus, the nuclear dynamic architecture, might explain the giant size and complex organization of primary transcripts and their introns.

The 33 kb transcript of the chicken alpha-globin gene domain is part of the nuclear matrix.

Giant nuclear transcripts, and in particular the RNAs of the globin gene domains which are much larger than their canonical pre-mRNAs, have been an enigma for many years. We show here that in avian erythroblastosis virus (AEV)-transformed chicken erythroleukaemic cells, where globin gene expression is abortive, the whole domain of alpha-globin genes is transcribed for about 33 kb in the globin direction and that this RNA is part of the nuclear matrix. Northern blot hybridisation with strand-specific riboprobes, recognising genes and intergenic sequences, and RT-PCR with downstream primers, show that the continuous full domain transcript (FDT) starts in the vicinity of a putative LCR and includes all the genes as well as known regulatory sites, the replication origin, and the DNA loop anchorage region in the upstream area. Absent in chicken fibroblasts, the globin FDT overlaps the major part of the ggPRX housekeeping gene that is transcribed in the opposite direction. RT-PCR and in situ hybridisation with genic and extra-genic globin probes demonstrated that the globin FDT is a component of the nuclear matrix. We suggest that the globin FDTs keep the domain in an active state, and the globin RNAs on the processing pathway are a component of the nuclear matrix. They may take part in the dynamic nuclear architecture when productively processed, or turn over slowly when globins are not synthesised.

Historical review: the discovery of 'giant' RNA and RNA processing: 40 years of enigma.

RNA processing is a primordial paradigm of gene expression. Iconoclastic when discovered, after 40 years there is still no general rationale for this apparent 'wasting' of up to 90% of RNA transcripts. This article tells the story of the discovery of RNA in the laboratory of J.E. Darnell. The discovery of 'giant' RNA and its conversion into rRNA revealed the phenomenon of RNA processing and pre-rRNA. Genuine mRNA was also identified, but the majority of DNA-like nuclear RNA was also found to be giant and unstable. In spite of early evidence, pre-mRNA processing was only accepted in 1977 when the discovery of gene fragmentation in DNA made it obvious.

Identification of an intronic TG repeat polymorphism in the human proteasome core particle PROS-27K gene.

A TG dinucleotide repeat was identified in intron 6 of the human proteasome core particle PROS-27K (IOTA, PSMA6) gene. We present data on the length polymorphism of this repeat in 120 individuals from Latvia and 197 individuals from Finland. A combination of PCR and fluorescent gel electrophoresis was utilized to type the polymorphism. Twelve alleles were observed, varying in length from 10 to 23 TG repeats. Similar allele frequencies were observed in Latvian and Finnish subjects, with 17 and 20 repeats being the most frequent in both populations. We suggest that this TG dinucleotide repeat could be utilized as a prospective marker for genetic linkage and association studies of common diseases.