An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA–protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model.     

Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.     

Minor contribution of an internal ribosome entry site in the 5'-UTR of ornithine decarboxylase mRNA on its translation

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5′-untranslated region (5′-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5′-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m⁷GpppG). Rapamycin inhibited ODC synthesis by 40-50% at both the G₁/S boundary and the G₂/M phase. These results indicate that an IRES in the 5′-UTR of ODC mRNA does not function effectively.     

The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m⁷GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions

The mature 3′-end of many chloroplast mRNAs is generated by the processing of the 3′-untranslated region (3′-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3′-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3′-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3′-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3′-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3′-UTRs, finding that each 3′-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3′-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3′-UTR processing activity on ortholog spinach 3′-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3′-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3′-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.     

Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis.     

Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.     

Single molecule measurements of titin elasticity

Titin, with a massive single chain of 3--4MDa and multiple modular motifs, spans the half-sarcomere of skeletal and cardiac muscles and serves important, multifaceted functions. In recent years, titin has become a favored subject of single molecule observations by atomic force microscopy (AFM) and laser optical trap (LOT). Here we review these single titin molecule extension studies with an emphasis on understanding their relevance to titin elasticity in muscle function. Some fundamental aspects of the methods for single titin molecule investigations, including the application of dynamic force, the elasticity models for filamentous titin motifs, the technical foundations and calibrations of AFM and LOT, and titin sample preparations are provided. A chronological review of major publications on recent single titin extension observations is presented. This is followed by summary evaluations of titin domain folding/unfolding results and of elastic properties of filamentous titin motifs. Implications of these single titin measurements for muscle physiology/pathology are discussed and forthcoming advances in single titin studies are anticipated.     

Calcium-dependent inhibition of in vitro thin-filament motility by native titin

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show — using in vitro motility and binding assays — that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin-thin filament interaction occurs in a calcium-dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.     

Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3′-untranslated region

Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3′-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3′-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3′-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3′-UTRs cloned 3′ to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.     

The 5' UTR of protein kinase C epsilon confers translational regulation in vitro and in vivo

We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells.