Structural aspects of microRNA biogenesis

One of the biggest surprises at the beginning of the 'post-genome era' was the discovery of numerous genes encoding microRNAs. They were found in genomes of such diverse organisms as Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens which implies their important role in multicellular life evolution. The number of microRNA genes is estimated to be nearly 1% of that of protein-coding genes. Their products, tiny RNAs, are thought to regulate gene expression during development, organogenesis, and very likely during many other processes, by hybridizing to their target mRNAs. The cellular functions of mRNAs that are regulated by microRNAs are only beginning to be revealed, and details of this regulation mechanism are still poorly understood. In this article we discuss the possible mechanisms of microRNA biogenesis with special emphasis on their structural aspects. We have focused on the factors and effects that may be responsible for the existing length differences between different microRNAs, and for the observed length heterogeneity within some individual microRNA species.     

Structural aspects of hydroxyproline-containing proteins

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Structural aspects of gas exchange

The lung is composed of several million small air spaces, lined by a delicate tissue membrane separating air from capillary blood. The design features of the gas exchange region in the lung are optimal for gaseous diffusion, by having a very extensive contact surface but with a minimal tissue barrier composed of an epithelial and endothelial layer separating an interstitial layer. The extent of the gas exchange surface in adult lungs is determined by general maturation which in turn is influenced by metabolic requirements of the organism. Environmental factors can modulate the pattern of ultimate lung development. Lung inflation causes air spaces to expand mainly by a process of tissue unfolding beneath an extremely thin layer of alveolar surfactant. This ensures cellular integrity during extreme deformations while at the same time providing a reserve of gas exchange surface so that functional diffusion capacity at all lung volumes is less than the structural maximum.     

Structural aspects of ganglioside-containing membranes

The demand for understanding the physical role of gangliosides in membranes is pressing, due to the high number of diverse and crucial biological functions in which they are involved, needing a unifying thread. To this purpose, model systems including gangliosides have been subject of extensive structural studies. Although showing different levels of complication, all models share the need for simplicity, in order to allow for physico-chemical clarity, so they keep far from the extreme complexity of the true biological systems. Nonetheless, as widely agreed, they provide a basic hint on the structural contribution specific molecules can pay to the complex aggregate. This topic we address in the present review. Gangliosides are likely to play their physical role through metamorphism, cooperativity and demixing, that is, they tend to segregate and identify regions where they can dictate and modulate the geometry and the topology of the structure, and its mechanical properties. Strong three-dimensional organisation and cooperativity are exploited to scale up the local arrangement hierarchically from the nano- to the mesoscale, influencing the overall morphology of the structure.     

Structural aspects of proton-pumping ATPases

ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1 a1b2c10-12. The first five of these constitute a globular structure, F1-ATPase, which is bound to an intrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypeptides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequence of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, delta mu H+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, F1-ATPase. They diffract to a resolution of 3-4 A and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles.     

Structural and functional aspects of stomata

Differential cell wall thickening in developing guard cells of Polypodium vulgare L. has been studied with particular reference to guard cell protoplast deformation and the eventual formation of the stomatal pore. Concomitant studies on the development of guard cell chloroplasts and their starch inclusions during ontogeny of the stomatal complex have provided data which have been incorporated into a model to account for the formation of the pore. Guard cell starch inclusions reach a maximum density per unit volume at the same time as the guard cell walls achieve maximum differential thickening. These events coincide with the development of the pore. It is suggested that, whilst pore formation is initiated enzymatically, the mechanical forces required to bring about the separation of the two guard cells are of an osmotic nature derived from starch hydrolysis. The development of the mesophyll in relation to the epidermis is examined in respect of the formation of substomatal chambers.     

Structural aspects of thrombin specificity.

Computer-assisted comparisons were made of the X-ray coordinates of all homologous atoms in the serine protease derivatives tosyl chymotrypsin Aα, tosyl elastase, and diisopropylphosphoryl trypsin. The results provided further quantitative support for the belief that sequence homology in proteins results in close similarity of conformation. On this basis, inferences were drawn about the three-dimensional structure of the serine protease thrombin, for which atomic coordinates have not yet been determined experimentally. Further, it was concluded that the unique specificity of thrombin, i.e., its selective cleavage of certain ArgGly bonds in fibrinogen, is unlikely to be due to the insertions in the amino acid sequence of thrombin or to differences in sequence in the region of the active site and binding pocket. It is possible, however, that the elongated A chain appended to thrombin may be a source of this specificity.