Analysis of the message-sequence content of the pulse-labeled poly(A)+ heterogeneous nuclear RNA from HeLa cells by cDNA-excess hybridizations.

The message-sequence content of pulse-labeled poly(A)+ HeLa heterogenous nuclear RNA (hnRNA) has been examined by hybridizations to an excess of message cDNA. Control experiments show that the message cDNA accurately reflects the sequence distribution of the complex mixture of poly(A)+ messages present in the HeLa cytoplasm. Pulse-labeled poly(A)+ molecules in both the lamina-associated and shnRNA fractions contain message sequences, and approximately 65% of the poly(A)-adjacent hnRNA sequences are homologous to the 3' ends of mRNA. The majority of the pulse-labeled hnRNA molecules contain abundant message sequences. By use of these techniques it is also shown that some pulse-labeled polyadenylated message sequences are still synthesized in the presence of the adenosine analogue 5,6-dichloro-beta-D-ribofuranosylbenzimidazole under conditions where little or no new cytoplasmic mRNA is produced.     

Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids

The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.     

Deadenylylation: a mechanism controlling c-fos mRNA decay

The c-fos proto-oncogene mRNA is extremely labile and is rapidly degraded within minutes after being transported to the cytoplasm of growth factor-stimulated fibroblasts. Analysis of the structural determinants controlling c-fos message decay reveals that this message contains at least two functionally independent elements that are responsible for its short half-life. One of these determinants is an AU-rich sequence present in the 3' untranslated region of the c-fos message, whereas the other determinant, which is structurally unrelated to the AU-rich element, is located within the c-fos protein-coding sequence. Both the c-fos AU-rich element and the coding region instability determinant appear to function by facilitating rapid removal of the c-fos poly(A) tail as a first step in the mRNA degradation process.     

Calf lens crystallin messenger RNA''s contain polynucleotide sequences rich in adenylic acid

Calf lens messenger fractions have been purified on poly(dT)-cellulose. The purified RNA's have been analyzed as nucleosides by a method which can be used on a micro-scale with highly reproduceble results. Both the 10S and 14S lens mRNA contain poly A sequences. The poly A content found in 14S mRNA cannot account for the excess nucleotides in this message.     

Construction of a representative cDNA library from mRNA isolated from mouse oocytes

A representative cDNA library has been constructed from the small quantities of poly(A)+ RNA present in unfertilised mouse oocytes. The construction of this library has been achieved by use of cow pea mosaic virus RNA as a carrier during isolation of polyadenylated message and during subsequent cloning procedures. This approach may be applicable to any system in which amounts of mRNA are limiting.     

The sequence of porcine αs2-casein cDNA

cDNA clones encoding the entire porcine alpha s2-casein message were isolated and sequenced. The porcine alpha s2-casein cDNA is 1093 bp, excluding the poly(A) tail, in length and encodes a preprotein of 235 amino acids.     

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3''-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.     

Why,when and how does the poly(A) tail shorten during mRNA translation?

• 1.1. The length of the poly(A) tail at the 3'-end of mRNA may control protein synthesis by bringing the 3'-end in close proximity to the 5'-end of the noncoding region as well as increasing the duration of mRNA translation by its binding to the poly(A) binding protein.
• 2.2. The rate-limiting step in the decay of the body of the message is the shortening of a long poly(A) tail during mRNA translation. The shortening of the poly(A) tail occurs during pre-elongation in the protein synthesis cycle.
• 3.3. The shortening of the poly(A) tail during mRNA translation may not involve RNase activity, however poly(A) binding protein seems to play a role, at least in part, in shortening of the poly(A) tail.

     

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.     

Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: Reversal upon cell attachment to a surface

Anchorage-dependent cells, when forced into suspension culture, display a repertoire of dramatic, coordinated regulatory phenomena. Message production promptly decreases 5 fold but the cells maintain a constant amount of poly(A)⁺ by means of a concomitant stabilization of mRNA against decay. Protein synthesis shuts down much later and the mRNA is stored in a nonfunctioning state. In this study, the inactive mRNA is extracted from suspended cells and shown to have aberrant translation properties. Well defined polypeptides are apparently no longer synthesized when this mRNA directs protein formation in either reticulocyte or wheat germ-derived heterologous translation systems. Rather, shortened peptides are formed by this mRNA and these become smaller as mRNA is used from cells suspended for longer periods of time. Very few focused spots are formed when the aberrant polypeptides are analyzed in two-dimensional electrophoresis.The sedimentation properties of suspended cell mRNA and the size of poly(A) are unchanged from control monolayer cells. Cross-hybridization of cDNA transcribed from a control cell message population with suspended cell mRNA shows that all sequences are present in normal concentrations. While most identifiable spots disappear from the two-dimensional gel electropherograms of the protein products produced by suspended cell mRNA, a few polypeptides are still synthesized in relatively normal amounts. Conserved polypeptides are found in products of both the reticulocyte and wheat germ systems, but they are different products in each case. The lesion in the suspended cell mRNA does not seem to be at the 5′ termini, since synthesis of the shortened peptides is fully sensitive to inhibition by pm⁷G.Cells that contain extensively modified message can resume protein synthesis when allowed to reattach to a solid substrate. There is an apparent remodification of mRNA to normal translatability within a few hours of cell reattachment, since mRNA from recovering cells quickly resumes directing relatively normal patterns of polypeptide synthesis in vitro. The restoration of normal message function occurs even when new message formation is blocked with actinomycin.Cells recovering after reattachment synthesize supranormal amounts of a few major proteins involved with cell structure, as shown in these studies by an increased amount of translatable sequences which encode these proteins. The most apparent enhanced message is that coding for actin. mRNA from recovering cells produces in vitro several times more actin relative to other proteins than does control cell mRNA. The enhancement of actin mRNA is not seen in the message population of cells that reattach in the presence of actinomycin. The results suggest a morphologically related induction of gene expression.     

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

Identification of two cDNA clones coding for androgen-dependent polypeptides in rat ventral prostate

cDNA clones were obtained by transformation of $\text{E. coli}$ x1776 with pBR322 containing insert of ds cDNA synthesized from total rat prostate poly(A) RNA. Two prostate-specific cDNA clones were isolated by colony hybridization and identified by message selection/translation as encoding polypeptides of M_r: 13,500 and 9,300. Hybridization of poly(A) RNA from normal and castrated rat prostates to the cloned cDNAs indicated that the levels of mRNAs coding for M_r: 13,500 and 9,300 polypeptides are regulated by testosterone.     

Poly(U)-dependent polyphenylalanine and polytyrosine synthesis in vitro by a tRNATyr variant with an enzymatically altered anticodon, G-A-A

A variant of T. utilis tRNATyr containing a base substitution (psi----A) in the middle position of the anticodon has been constructed by enzymatic procedures in vitro. This variant is unique in that it can accept both tyrosine and phenylalanine. This tRNA was shown to be active in transferring both tyrosine and phenylalanine into polypeptides in a cell-free, poly (U)-directed translation system from yeast. This result gives further support to the adapter hypothesis since tyrosine, attached to the variant tRNATyr with an anticodon G-A-A, is incorporated into polypeptides in response to poly (U) message.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.