Messenger RNA in HeLa Cells:

Abstract

Messenger RNA (mRNA) and messenger like RNA (mlRNA) were investigated in the cytoplasm of HeLa cells while ribosomal RNA synthesis was arrested. Under these conditions, functional mRNA associated in polyribosomes and cytoplasmic free mlRNA are formed and can be labelled selectively to steady state.All cytoplasmic non-ribosomal RNA sedimenting a t more than 6-7 S exists in the form of ribonucleoprotein complexes which pre-exist in the cell, and are stable upon cell lysis, sedimentation and (after fixation) CsCl density gradient analysis. The functional, true mRNA is contained in a complex of mRNA and protein which bands in association with ribosomes (e = 1.52 to 1.60 g/cm3) in CsCl density gradients or, released by EDTA, a t its own intrinsic density of 1.40-1.88 g/cm3. The cytoplasmic free mlRNA bands as a particle of mlRNA-containing ribonucleoprotein at an identical low density. The molecular weight spectrum of mRNA is identical t o that of mlRNA and the sedimentation pattern of the mRNA-protein complex released from polyribosomes is similar to that of the free mlRNA -protein complex.The physico-chemical separation of mRNA -and mlRNA -protein complexes allowed us to follow their relative kinetics of synthesis and decay. Each type of ribonucleoprotein obeys a different, strictly time-dependent pattern. Label enters the pool of free mlRNA -protein complexes first and may, in a pulse-chase experiment, be partially chased into polyribosomes. At steady state (6 h) 40-60°/, of the labelled RNA remains in the form of free mlRNA-protein particles. These cannot be chased into polyribosomes, the kinetics of mRNA. and mlRNA-protein complexes decay following identical patterns. These findings are in agreement with a model according to which mlRNA from the nucleus first joins the pool of free ribonucleoprotein. Then, the activated mRNA-protein complexes attach to ribosomal subunits and form polyribosomes whereas inactivated mlRNA * protein complexes remain free in the cytoplasm.In order to further strengthen the evidence in favour of the real existence of mRNA-and mlRNA * protein complexes in the cells, the corresponding fractions from sedimentation or CsCl density gradients were observed in the electron microscope. By this method it was possible to see small cytoplasmic particles which have not before been identified. The rounded structures, with diameters ranging from 100 h to 200 8, seem to consist of the coiling of a 35 8 wide pearllike chain which may also be identsed in polyribosomes. The frequency of these particles is highest in the mlRNA. protein band (e = 1.40-1.48 g/cmS). Thus they may correspond to the mRNA -protein complex. However since they share some morphological features with other known biological structures the evidence is not conclusive.t We are desolated to announce to our friends and collegues that Nicole Granbouland died in an accident shortly before the completion of this manuscript. We dedicate this paper to her memory.Unusuul Abbreviations. mRNA, messenger RNA (this term is restricted to function...

Dissociation and Reassociation of Globin‐Synthesizing Polyribosomes from Immature Avian Red Cells

“…The pellet containing mostly polyribosomes was suspended by gentle homogenisation in suspension buffer at a concentration of 10 A,,, units per ml. The upper third of the 40-min supernatant was recentrifuged in the Ti-60 rotor a t 60000rev./min for a further 140 min to remove ribosomal material and free messenger-like RNA * protein complexes [8] and served in the cell-free systems as the source of enzymes and tRNAs (180-min supernatant).…”

“…Aliquots were fixed with formaldehyde according to Spohr [8] and analyzed on glycerol gradients. The magnesium concentration of the remaining unfixed material was restored by overnight dialysis against suspension buffer containing 2 mM magnesium.…”

The protein of Mr 21 000 constituting the prosome‐like particle of duck erythroblasts is homologous to apoferritin

“…Extensive investigations carried out in several laboratories over the last decades showed that mRNA exists in essentially two compartments of the cytoplasm; the translated mRNA in polyribosomes which is associated with a variety of translational-control factors forming an mRNP (m, messenger) upon dissociation of the ribosomes and the non-translated repressed mRNA associated with another set of protein and RNP factors, which keep it in a non-translatable status zn vitro and, most likely, in vivo (Spohr et al, 1970). The former mRNP includes ubiquitous factors common to all types of mRNA [cap-binding and poly(A)-binding proteins, initiation factors, etc.…”

Adenosine‐rich sequences in rapidly hybridizing messenger‐like RNA and their possible significance for reiterated base sequences in eukaryotic DNA

“…Duck erythroblasts and HeLa cells were labelled with 32P or 3H-uridine and RNA was extracted, purified and fractionated as previously described [3,13,191. Hybridization was carried out either in liquid phase and evaluated by the Sephadex technique [3] or on filters by the urea low-temperature technique [2] .…”