Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin

The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease.

Brief digestion of HeLa nuclei with mirococcal nuclease releases monomer hnRNP particles as well as monomer and polynucleosomes. Sucrose gradient analysis of the nuclease released material reveals a series of small A260 peaks overlapping a more predominant peak in the 40S region of the gradient. Analysis of the proteins, DNa, and RNA in successive gradient fractions has confirmed that the smaller peaks are monomer and polynucleosomes, and that the larger peak is 40S hnRNP. Like 40S particles isolated by low salt extraction or by sonication, the nuclease released particles are composed of rapidly labeled RNA associated with a group of non-histone proteins the most predominant of which are the 32,000-44,000 MW proteins previously identified as core hnRNP proteins. These results provide further evidence that 40S hnRNP particles exist as discrete structural components of larger in vivo ribonucleoprotein complexes

Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles.

Partial acid cleavage, comparative HPLC tryptic peptide mapping and amino acid sequencing of the C1 and C2 proteins of HeLa heterogeneous nuclear ribonucleoprotein (hnRNP) particles demonstrate that proteins C1 and C2 differ in primary structure by the presence of a 13 amino acid insert sequence in C2. This C2 insert sequence occurs after either glycine 106 or serine 107 in C1. The additional 13 amino acids that are present in C2 account for the observed molecular weight difference between the C1 and C2 hnRNP proteins on SDS polyacrylamide gel electrophoresis. Because C1 and C2 appear identical except for the 13 residue insert and because the 3' and 5' untranslated regions of the corresponding mRNAs also appear to be the same (Swanson et al., Mol. Cell. Biol. 7: 1731-1739), it is possible that both polypeptides are produced from a single transcription unit through an alternative splicing mechanism

Oligonucleotide binding specificities of the hnRNP C protein tetramer.

Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3' intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3' splice site, IVS2 of human alpha-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20were used as competitors. A 20 nt beta-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines