Maternal mRNA expression in early development: regulation at the 3' end

Early development in many animals is programmed by maternal mRNAs inherited by the fertilized egg. Many of these RNAs are translationally dormant in immature oocytes, but are recruited onto polysomes during meiotic maturation or fertilization. Polyadenylation plays a major role in controlling the translation of maternal mRNA during these times of development. Polyadenylation, in turn, is dependent upon two cis elements that reside in the 3'-terminal region of responsive mRNAs. In two cases, the factors that interact with these regions have been examined. The half-life of maternal mRNA also is regulated by polyadenylation, which again is controlled by 3'-terminal cis elements. The recent literature covering these topics is reviewed.     

Novel integration sites at the distal 3' end of the c-myb locus in retrovirus-induced promonocytic leukemias.

In BALB/c nu/nu and sublethally irradiated DBA/2 mice, promonocytic leukemia was induced by intravenous inoculation of Friend murine leukemia virus (F-MuLV) strain C57 in conjunction with intraperitoneal injection of pristane. These tumors appear to be identical morphologically to previously reported ones induced by other MuLVs, such as Moloney, amphotropic 4070A, and F-MuLV FB29, which most commonly have provirus integrations in the 5' end of the c-myb locus. Interestingly, 2 of the 16 F-MuLV-induced tumors had viruses integrated in the distal 3' end of c-myb. To determine the precise locations of these integrations, it was necessary to clone sequences encoding the 3' c-myb exons and to prepare a physical map of this region. Exons 10 to 15 were positioned on the map, and it was found that the proviruses in the aforementioned tumors were located within narrow region in the beginning of the large (greater than 11 kb) intron 14. The predicted protein product encoded by the affected alleles is truncated by 38 amino acids. This represents a novel virus integration site which is most likely associated with oncogenic activation of the c-myb gene during leukemogenesis.     

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.     

The stem-loop structure at the 3'' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.     

Sequence analysis of a KpnI family member near the 3' end of human beta-globin gene.

We determined the complete nucleotide sequence (6125 bp) of a full-length member of human KpnI family, designated T beta G41, which is located about 3 kb downstream from the beta-globin gene. Comparison of the sequence with the KpnI family sequence compiled by Singer revealed that a new 131 bp sequence is present in the T beta G41. Hybridization analyses showed that a few thousand of human KpnI family members are carrying this additional sequence. Computer search of DNA databases for T beta G41-homologous sequence showed that some T beta G41-homologous sequences were closely associated with pseudogenes. The T beta G41 sequence also showed significant sequence homology with ChBlym-1, a transferrin-like transforming gene of chicken. Furthermore, an amino acid sequence deduced from the T beta G41 nucleotide sequence revealed a relatively-high homology to those of human transferrin and lactotransferrin.     

Changes in allele‐specific association of histone modifications at the imprinting control regions during mouse preimplantation development

Allele‐specific association of histone modification is observed at the regulatory region of imprinted genes and has been suggested to work as an epigenetic marker for monoallelic gene expression, along with the allelic CpG methylation of DNA. Although the parent‐origin‐specific epigenetic status in imprinted genes is thought to be established during preimplantation development, little is known about the allelic specificity of histone modifications during this period because of the limited volume of material available for analysis. In this study, we first revealed the allelic enrichment of histone modifications and variant histones at the imprinting control regions (ICRs) of four‐cell to blastocyst stage preimplantation embryos by using carrier chromatin immunoprecipitation and sequence polymorphism analysis of immunoprecipitated DNA. We found relative enrichment of histone H3 lysine 9 dimethylation at the imprinted alleles of ICRs and obtained the results suggesting that histone modifications at ICRs are established during a late preimplantation stage. genesis, 47:611–616, 2009. © 2009 Wiley‐Liss, Inc.     

Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.     

Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.

The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.     

Identification of a novel sequence at the 3' end of the GB virus B genome

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.