Klaus Scherrer

Proteins associated with globin messenger RNA in avian erythroblasts: Isolation and comparison with the proteins bound to nuclear messenger-likie RNA.

There is evidence that the polyribosomal messenger RNA (mRNA) in animal cells is specifically associated with proteins [l-7] . In the case of cytoplasmic [5,8] or nuclear [9] messenger-like RNA (mlRNA) similar RNA-protein (RNP) complexes have also been described. Contrary to the claim that such associations may be non-specific [ 10, 1 l] , adequate control experiments suggest that they pre-exist in the cell and are not artefacts produced during lysis [5,8, 121. The exact physiological significance of these structures is not known. They may play a variety of roles such as: protection against RNase attack [5,8], RNA transport [ 1,8,9] , cleavage of giant mlRNA and selection of the RNA molecules to be transferred to the cytoplasm [5,9] , or initiation of protein synthesis [7] . In particular, two reports [ 13, 141 claim that the protein bound to the giant nuclear mlRNA is the same as that associated with mRNA in polyribosomes, indicating a possible function in mRNA transport. We have studied these mRNP particles in a highly differentiated system: duck immature red blood cells. In contrast to the hemoglobin producing cells in mammals, duck red cells are nucleated, making possible the investigation of the mland mRNA complexes in the nucleus and in the cytoplasm of a cell where a specific mRNA can be identified.

Evidence for a translational inhibitor linked to globin mRNA in untranslated free cytoplasmic messenger ribonucleoprotein complexes

In eukaryotic cells, mRNA exists in the cytoplasm in the form of ribonucleoprotein complexes [l-3]. In the case of duck immature erythrocytes, the RNAsequences coding for globin chains have been localized in two sub-cytoplasmic fractions, the ‘15 S mRNP bound in polyribosomes and the ‘20 S’ mRNP free in the cytoplasm [4-61. The proteins bound to globin mRNA from both cytoplasmic sources were previously shown to differ substantially in their composition [S] , and it is therefore theoretically possible that the proteins associated with a given mRNA molecule can determine its localization and function within the cytoplasm. The purpose of this paper is to investigate the possible role of the constituents bound to globin mRNA in the process of translation by analysing, in vitro, the translational activity of the two mRNPs.

Mapping of structural and transcription-related matrix attachment sites in the alpha-globin gene domain of avian erythroblasts and erythrocytes.

The positions of preferential DNA interaction with the nuclear matrix were mapped within the domain of the chicken alpha-globin genes in transcriptionally active erythroblast nuclei and inactive nuclei of mature erythrocytes. In the latter, only two major distinct attachment sites were observed, close to the A + T-rich sequences previously found at the boundaries of the domain. Sequencing of these structural matrix attachment points revealed several known DNA motifs; some of them were present on both sides of the domain. In actively transcribing erythroblast nuclei of adult animals, a large fraction of the transcribed area was represented in nuclear matrix DNA, including upstream and downstream elements. In particular, adult alpha A- and alpha D-globin genes were found in matrix DNA, while the transcribed but translationally unexpressed embryonic pi gene was underrepresented. The data are discussed in terms of the existence of stable or structural and expression-related matrix attachment sites; correlations to the origin of replication and the units of transcription of the domain are shown.

Gene and genon concept: coding versus regulation

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 methylated constituents of globin mRNA

Recent findings indicate that messenger RNA from a wide variety of cellular and viral sources contains methylated constituents [l-5] . Studies of complex mixtures of mammalian mRNA’s have indicated that the methyl groups occur in internal residues of N6 methyl adenylate and in unusual sequences at the 5’-termini [S-l 11. The 5’-terminal sequences appear to be of the form X5’ ppp” (Nmp) 1-2N~ . . . in which a ‘capping nucleoside’ X (generally m’G), is joined by a 5’-5’ triphosphate linkage to sequences containing either one or two 2’0ribose methylated nucleotides (Nmp). In order to determine whether such components are also characteristic of a well-defined species of eukaryotic mRNA, we investigated the methylated derivatives of mRNA in immature duck erythrocytes. These cells synthesize readily detectable quantities of 9 S globin mRNA in addition to a variety of other mRNA’s [ 121. Our findings indicate that 9 S globin mRNA also contains capped 5’-terminal sequences, but very little, if any, internal base methylated derivatives. Moreover, compared to a mixture of other mRNA species synthesized by these cells, the 9 S mRNA is relatively enriched in the sequences that contain two 2’0methyl nucleotides.

Phylogenic relationships of the amino acid sequences of prosome (proteasome, MCP) subunits.

Prosomes [or proteasomes, Multi-Catalytic Proteinase (MCP)] are multisubunit protein complexes, found from archaebacteria to man, the structure of which (a 4-layer cylinder) is remarkably conserved. They were first observed as subcomplexes of untranslated mRNP, and then as a multicatalytic proteinase with several proteolytic activities. A number of sequences from subunits of these complexes are now available. Analysis of the sequences shows that these subunits are evolutionarily related, and reveals three highly conserved amino acid stretches. Based on a phylogenic approach, we propose to classify the sequenced subunits into 14 families, which fall into two superfamilies, of the α- and β-type. These data, together with several recently published observations, suggest that some subunits may be interchangeable within the complexes, which would thus constitute a population of heterogenous particles.

The Prosomes (Multicatalytic Proteinases; Proteasomes) and Their Relationship to the Untranslated Messenger Ribonucleoproteins, the Cytoskeleton, and Cell Differentiation†

Publisher Summary This chapter discusses the proteinase function only in relation to the prosome story as a whole and the enzymological mechanisms. Prosomes are “faculative” ribonucleoproteins of about 720,000 M r that display a multicatalytic proteinase activity. Prosomes, called multicatalytic proteinase (MCP) complexes or proteasomes, by many enzymologists, are a new type of cellular factor. They show up in the most varied and unexpected contexts of cellular structure and function, from archeobacteria to humans. They are not only MCPs, with highly selective substrate-specificity, but also a subcomplex of the untranslated messenger ribonucleoprotein (mRNP); they incorporate in a particular state a small ribonucleic acid (RNA) that, in the case of mammalian prosomes, turns out to be a reverse primer of the “retroviral” transfer RNA (tRNA) type. In addition, an important theoretical concept emerges: if the fundamental mechanisms of the mRNA translation are based on ubiquitous factors, the essential differential controls of specific mRNA expression in the cytoplasm are based on a system of mRNA stabilization, acting positively, and of negative controls, by trans-acting factors, exerting their effect on the untranslated mRNA.

Correlation of specific coding sequences with specific proteins associated in untranslated cytoplasmic messenger ribonucleoprotein complexes of duck er

Cytoplasmic messenger RNA exists in animal cells in the form of two types of mRNA-protein (mRNP) complexes: in polyribosomes containing the actively translated mRNA, and as free cytoplasmic mRNP with untranslated mRNA. In the case of globin mRNA, these complexes were isolated and purified as a ‘15 S’ particle from polyribosomes and as ‘20 S’ free mRNP; we found that these two different functional forms of globin mRNP contain two extensively different sets of accompanying proteins [ 11. Recently, we could demonstrate that the 20 S globin mRNA is untranslatable in vitro prior to deproteinization whereas, in contrast, the 15 S globin mRNP from polyribosomes is as readily translatable in vitro as the purified 9 S globin mRNA isolated from 20 S or 15 S particles-[2]. This

The nuclear matrix of duck erythroblasts is associated with globin mRNA coding sequences but not with the major proteins of 40S nuclear RNP

Abstract A residual protein matrix has been prepared from avian erythroblast nuclei by extensive extraction with salines and detergent and subsequent digestion with high concentrations of RNase and DNase. Ultrastructural examination reveals considerable internal structure, the most prominent feature being the remains of the nucleoli embedded in a network of fibres of fairly uniform diameter of 50 A. The proteins which make up this structure have been examined by two-dimensional electrophoresis and are shown to consist of a characteristic set of about 30, mainly acidic components, including four prominent species of 43 000, 52 000, 66 000 and 68 000 molecular weight (MW). In parallel preparations of the nuclear matrix digested with DNase alone, much of the nuclear RNA is found associated with the residual structure, including globin-coding sequences. These results correlate well with the ultrastructural appearance of DNase-digested matrix preparations which show that superimposed on the 50 A fibrous network is a 200–300 A granular component, the combined fibrillo-granular structure resembling the interchromatin RNP previously identified in situ. However, the proteins of the DNase-digested matrix seen by two-dimensional electrophoresis are indistinguishable from the proteins of matrix preparations digested with both DNase and RNase. Furthermore, two-dimensional comparison between the proteins of the DNase-digested matrix and purified 40S nuclear RNP particles shows that the bulk of the proteins found associated with nuclear RNA in vitro are extracted during matrix preparation, and only two, with MWs of 43 000 and 73 000, remain. The latter species co-migrates with the poly(A)-binding protein.

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.