Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation.

The highly unstable c-myc mRNA has been shown to be stabilized in cells treated with protein synthesis inhibitors. We have studied this phenomenon in an effort to gain more insight into the degradation pathway of this mRNA. Our results indicate that the stabilization of c-myc mRNA in the absence of translation can be fully explained by the inhibition of translation-dependent poly(A) tail shortening. This view is based on the following observations. First, the normally rapid shortening of the c-myc poly(A) tail was slowed down by a translation block. Second, c-myc messengers which carry a short poly(A) tail, as a result of prolonged actinomycin D or 3'-deoxyadenosine treatment, were not stabilized by the inhibition of translation. We propose that c-myc mRNA degradation proceeds in at least two steps. The first step is the shortening of long poly(A) tails. This step requires ongoing translation and thus is responsible for the delay in mRNA degradation observed in the presence of protein synthesis inhibitors. The second step involves rapid degradation of the body of the mRNA, possibly preceded by the removal of the short remainder of the poly(A) tail. This last step is independent of translation.     

Non-snRNP U1A levels decrease during mammalian B-cell differentiation and release the IgM secretory poly(A) site from repression

A regulated shift from the production of membrane to secretory forms of Immunoglobulin M (IgM) mRNA occurs during B cell differentiation due to the activation of an upstream secretory poly(A) site. U1A plays a key role in inhibiting the expression of the secretory poly(A) site by inhibiting both cleavage at the poly(A) site and subsequent poly(A) tail addition. However, how the inhibitory effect of U1A is alleviated in differentiated cells, which express the secretory poly(A) site, is not known. Using B cell lines representing different stages of B cell differentiation, we show that the amount of U1A available to inhibit the secretory poly(A) site is reduced in differentiated cells. Undifferentiated B cells have more total U1A than differentiated cells and a greater proportion of this is not associated with the U1snRNP. We show that this is available to inhibit poly(A) addition at the secretory poly(A) site using cold competitor RNA oligos to de-repress poly(A) addition in nuclear extracts from the respective cell lines. In addition, endogenous non-snRNP associated U1A-immunopurified from the different cell lines-inhibits poly(A) polymerase activity proportional to U1A recovered, suggesting that available U1A level alone is responsible for changes in its inhibitory effect at the secretory IgM poly (A) site.     

Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site

We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A. U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells. U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites. We therefore probed the structure surrounding the novel U1A binding sites. We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site. The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites. However, long range interactions are responsible for maintaining them in an open configuration. Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5' splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site. These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA. This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice.

The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.     

Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the post-ribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.     

RNA Polyadenylation Sites on the Genomes of Microorganisms,Animals, and Plants

Pre–messenger RNA (mRNA) 3′-end cleavage and subsequent polyadenylation strongly regulate gene expression. In comparison with the upstream or downstream motifs, relatively little is known about the feature differences of polyadenylation [poly(A)] sites among major kingdoms. We suspect that the precise poly(A) sites are very selective, and we therefore mapped mRNA poly(A) sites on complete and nearly complete genomes using mRNA sequences available in the National Center for Biotechnology Information (NCBI) Nucleotide database. In this paper, we describe the mRNA nucleotide [i.e., the poly(A) tail attachment position] that is directly in attachment with the poly(A) tail and the pre-mRNA nucleotide [i.e., the poly(A) tail starting position] that corresponds to the first adenosine of the poly(A) tail in the 29 most-mapped species (2 fungi, 2 protists, 18 animals, and 7 plants). The most representative pre-mRNA dinucleotides covering these two positions were UA, CA, and GA in 17, 10, and 2 of the species, respectively. The pre-mRNA nucleotide at the poly(A) tail starting position was typically an adenosine [i.e., A-type poly(A) sites], sometimes a uridine, and occasionally a cytidine or guanosine. The order was U>C>G at the attachment position but A>>U>C≥G at the starting position. However, in comparison with the mRNA nucleotide composition (base composition), the poly(A) tail attachment position selected C over U in plants and both C and G over U in animals, in both A-type and non-A-type poly(A) sites. Animals, dicot plants, and monocot plants had clear differences in C/G ratios at the poly(A) tail attachment position of the non-A-type poly(A) sites. This study of poly(A) site evolution indicated that the two positions within poly(A) sites had distinct nucleotide compositions and were different among kingdoms.     

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.     

A mRNA determinant of gRNA-directed kinetoplastid editing

Several mitochondrial mRNAs of the kinetoplastid protozoa do not encode a functional open reading frame until they have been edited through the addition or deletion of U nucleotides at specific sites. Genetic information specifying the location and extent of editing is present on guide RNAs (gRNAs). The sequence adjacent to most mRNA editing sites has a high purine content which previously has been proposed to facilitate the editing reaction through base-pairing to a poly(U) tail at the 3′ end of the gRNA. We demonstrate here that gRNA binding alone is insufficient to create an editing site and that the mRNA sequence near an editing site is an additional determinant affecting the efficiency of the reaction.     

B-lineage regulated polyadenylation occurs on weak poly(A) sites regardless of sequence composition at the cleavage and downstream regions.

Early/memory and plasma B-cell lines and fibroblasts were analyzed for their ability to use a 5' proximal (variant) versus a 3' distal (constant) poly(A) site, in the absence of a competing splice, from a set of related constructs. The proximal:distal poly(A) site use (P:D ratio) of the resulting cytoplasmic poly(A)+ mRNA is a measure of poly(A) site strength. In this context the immunoglobulin gamma2b secretory-specific poly(A) site showed a P:D ratio of 1:1 in plasma cells, 0.43:1 in early/memory B-cells and an intermediate value in fibroblasts. Meanwhile, a construct with a proximal SV40 early-like poly(A) site produced mRNA with a P:D ratio of >>50:1 in all cell types. Alterations in the region downstream of the proximal poly(A) addition site and at the site itself resulted in changes in the P:D ratio. However, these poly(A) sites, all with a P:D ratio of < or = 5:1, were used most efficiently in plasma cells. Constructs totally devoid of immunoglobulin sequences, but containing heterologous poly(A) sites producing mRNA with P:D ratios of < or = 5:1, were also used more efficiently in plasma cells. We therefore conclude that weak poly(A) sites, regardless of sequence composition, are used more efficiently in plasma cells than in the other cell types.