. "Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome." (NAS Colloquium) Molecular Kinesis in Cellular Function and Plasticity. Washington, DC: The National Academies Press, 2002.
The following HTML text is provided to enhance online
readability. Many aspects of typography translate only awkwardly to HTML.
Please use the page image
as the authoritative form to ensure accuracy.
Colloquium on Molecular Kinesis in Cellular Function and Plasticity
Fig. 1. ELAV/Hu RRM proteins form distinct granules in cell body and dendrites of neurons. Rabbit polyclonal serum prepared against recombinant HuB was used to visualize ELAV/Hu proteins in isolated rat embryonal cortical neurons by using confocal microscopy (reprinted from ref. 11). Prebleed serum showed no appreciable fluorescence of any neuronal samples (10, 11). The granules containing Hu proteins (A) coalesced following treatment with puromycin (B) to disrupt translation. [Reproduced with permission from ref. 11 (Copyright 1998, J. Cell Sci.).]
porter 1 (12), NF-M (13), GAP43 (15), VEGF (16), c-fos (17–19), c-myc (unpublished results), TNF-a (19), GM-CSF (19), and tau (20). With the exception of NF-M mRNA (13), the binding of these mRNA targets to ELAV/Hu proteins has only been demonstrated when using in vitro methods.
Messenger RNAs Are Generally Inabundant and Unstable. The average number of any particular mRNA species present in a mammalian cell varies over a range from less than one to as many as 1,000. This is in contrast to the U1 snRNA that is present in approximately 1 million copies per cell. In human cells, an average of about six copies of each mRNA per cell has been approximated with very few genes having at steady state as high as 50 to 100 copies per cell (21). In yeast, this number is approximately an order of magnitude lower. It is striking that so few copies of each mRNA are maintained in the steady state, and this suggests that mRNAs are continuously supplied and destroyed during normal cell metabolism. It is likely that a constant flux of mRNA through the mRNP infrastructure provides agility to the gene expression program. In profiling the expression of mRNAs by using techniques like microarray analysis or Serial Analysis of Gene Expression (SAGE), the steady-state level of each mRNA can be quantitated (22, 23). However, these procedures do not distinguish translationally active messages from inactive messages, and the relative turnover rate of each message can significantly affect protein output (23). Furthermore, the organization of mRNAs into functional complexes may influence their state of expression.
The instability of many mRNAs in comparison to ribosomal RNAs, transfer RNAs, and small nuclear RNAs, as well as their inabundance, has made analysis of their in vivo-associated protein interactions particularly difficult. As a result, most of what is currently known about mRNA-protein interactions has been derived from in vitro binding experiments. Nonetheless, the stability of endogenous mRNAs has been studied by using a variety of analytical tools (24–26). The relative stability of mRNAs involved in various biological processes can be depicted
Fig. 2. The relative stability of some diverse cellular mRNAs.
on a time line along which the half lives of ERG mRNAs (such as protooncogene and cytokine transcripts) is as short as a few minutes, and housekeeping proteins like cytoskeletal components and histones have half-lives equivalent to one full cell cycle (Fig. 2). It is generally true that whereas mRNAs that encode highly abundant and stable housekeeping proteins appear to be stable themselves, mRNAs encoding many growth regulatory proteins are very unstable (25). This instability is presumably due to the powerful and possibly undesirable effects on normal cell growth and differentiation that these gene products can have. The necessity to retain tight control over growth stimulatory proteins begins at the level of transcription, but is usually maintained also at the posttranscriptional level. Short half-lives for mRNAs encoding growth factors such as c-fos or c-myc allow cells to retain tighter control at the level of transcription, and therefore, the final production of the protein can be regulated with greater precision (24–26). In keeping with this line of reasoning, the ERG (also known as the immediate early gene) products encode growth regulatory proteins, and include mRNAs with short half-lives. The ability of the ELAV/Hu proteins to bind certain AU-rich-region-containing ERG mRNAs suggested to us that a large target set of AU-richregion-containing mRNAs might be captured by using ELAV/Hu proteins to identify en masse a unique subset of the total cell mRNA population (3). More recently, a direct in vivo approach has been possible by isolating mRNP complexes and identifying the mRNA subsets by using nucleic acid hybridization (1).
The Heterogeneous Nature of mRNPs. Heterogeneous nuclear RNA (hnRNA) and the correspondingly diverse hnRNPs have been recognized for many years (reviewed in refs. 27–30). Analysis on density and velocity gradients has revealed that whole-cell mRNA, and mRNA-binding proteins, often spread across a gradient making it difficult to discern specific proteins or mRNAs that might associate with one another (Fig. 3). This heterogeneity has caused the field to rely heavily on in vitro binding methods for study of the interactions between individual proteins and the sequence elements found in mRNAs (30, 31). It has been possible to analyze the migration of individual mRNAs on sucrose velocity gradients by using Northern blotting of gradient fractions. Indeed, several studies have used gradient analysis to localize translationally engaged mRNAs in fractions containing active polysomes (12, 13, 32, 33). However, mRNPs that are not associated with the assembled translation apparatus often remain widely distributed between the free mRNA and the assembled polysomes as exemplified with ELAV/Hu proteins (Fig. 3A). It has been assumed that these widely distributed mRNPs represent complexes containing mRNAs that are competent for, but not engaged in, translation. However, as shown in Fig. 3, treatment of cell extracts with EDTA can release the ELAV/Hu proteins from the region of active translation (ß complexes) and shift them to an intermediate position (a complexes). Similar results were evident when cells were treated with puromycin to inhibit translation without disrupting polysomes (Fig. 3C). As described by Tenenbaum et al. (1), mRNP complexes that are recovered by immunoprecipitation of tagged HuB from transfected P19 cells following treatment with EDTA