Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3'-untranslated region position-dependent cis-element and an untranslated region-binding protein

The genome of Trypanosoma cruzi contains tandem arrays of alternating genes encoding amastin and tuzin. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the protozoan parasite, and tuzin is a G-like protein. We demonstrated previously that the amastin-tuzin gene cluster is polycistronically transcribed to an equal extent in all parasite life cycle stages. The steady state level of amastin mRNA, however, is 68-fold more abundant in amastigotes than in epimastigotes. Here we show that the half-life of amastin mRNA is 7 times longer in amastigotes than in epimastigotes. Linker replacement experiments demonstrate that the middle one-third of the 630-nucleotide 3'-untranslated region (UTR) is responsible for the amastin mRNA up-regulation. This positive effect is dependent on the distance of the 3'-UTR segment from the stop codon and the polyadenylation site as well as on its orientation. A protein or protein complex more abundant in amastigotes than in epimastigotes binds to this minimally defined 3'-UTR segment and may be involved in its regulatory function.     

Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.     

UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation

Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3'-UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs.     

Possible Cis-acting signal that could be involved in the localization of different mRNAs in neuronal axons

Background  

Messenger RNA (mRNA) comprises three major parts: a 5'-UTR (UnTranslated Region), a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g.) neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs) found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region) in the 3'-UTR.     

Distribution and molecular characterization of mRNA-binding proteins specific to the (U)15 region of 3' UTR of the mouse catalase (Cas-1).

The 3' UTR of the mouse Cas-1 mRNA, encoding the antioxidant enzyme catalase, has a U-rich motif that is conserved across species. This motif is an active site for complex and dynamic interactions involving RNA-binding proteins. The spatial, temporal, and phylogenetic distribution of the Cas-1 3'-UTR U-rich motif-specific RNA-binding proteins was evaluated by gel mobility shift and UV cross-linking assays. The specific RNA-protein complexes were observed in mouse tissue homogenates representing developmental stages as early as day 10 pc and ranged in molecular weight from approximately 38 kDa to approximately 52 kDa. These mRNA-protein complexes appeared in all vertebrate species examined (human, mouse, rat, dog, rabbit, chicken, fish, and frog) but not in insects. The approximately 38-kDa protein was the most prominent protein in vertebrates. The cDNA sequence of the mouse approximately 38-kDa protein was obtained by purification of the protein, microsequencing, and RT-PCR. The resulting 456-nt sequence, representing the partial internal cDNA sequence, and its deduced amino acid sequence were similar to the RNA recognition motif (RRM) of a protein superfamily, implicated in splicing, stability, localization, and translation of RNAs. Although the results suggest that cis element-binding activity could be a cytoplasmic regulator of Cas-1 mRNA metabolism, the significance of this binding remains to be determined.     

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.