JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features

It is well known that non-coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high- and low-abundant proteins differ in the 5′ untranslated regions (UTR). Statistically, 5′ UTRs of low-expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5′ UTRs of high-expression mRNAs. Apart from the differences in 5′ UTRs, high-expression mRNAs contain stronger termination signals. Structural features of low- and high-expression mRNAs are likely to contribute to the yield of their protein products.     

Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Membrane-bound mRNAs are recruited from preinitiated ribonucleoprotein particles in injected Xenopus oocytes

Messenger RNA injected Xenopus oocytes exhibit a differential capacity for translation. mRNAs translated in the free cytoplasm are translated efficiently whereas mRNAs translated on the rough endoplasmic reticulum (RER) membrane are translated inefficiently. If mRNA injected oocytes are injected additionally with proteins isolated from the RER, enhanced translation of RER-bound mRNAs is observed. When examined by sucrose gradient centrifugation and RNA dot blots, most of the injected RER-bound mRNA sediments less than or equal to the 80 S monosome. The RER proteins recruit these preinitiated mRNAs onto polysomes as evidenced by a shift in sedimentation to the polysome region of a sucrose gradient. When examined by immunoblotting, the RER proteins are shown to contain a protein which reacts specifically with an antibody directed against docking protein (SRP-receptor protein). However, this putative docking protein does not appear to be the protein which actually recruits the preinitiated mRNAs onto polysomes.     

Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.

Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. We have used RNA selection to demonstrate that the FMRP RGG box binds intramolecular G quartets. This data allowed us to identify mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation.     

The destabilization of IL-2 mRNA by a premature stop codon and its differential stabilization by trans-acting inhibitors of protein synthesis do not support a role for active translation in mRNA stability.

To investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.     

General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells

The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors, including microRNAs (miRNAs). While it is well established that translation and mRNA degradation are tightly coupled, it is still not entirely clear where in the cell mRNA degradation takes place. In this study, we investigated the possibility of mRNA degradation on the ribosome in Drosophila cells. Using polysome profiles and ribosome affinity purification, we could demonstrate the copurification of various deadenylation and decapping factors with ribosome complexes. Also, AGO1 and GW182, two key factors in the miRNA-mediated mRNA degradation pathway, were associated with ribosome complexes. Their copurification was dependent on intact mRNAs, suggesting the association of these factors with the mRNA rather than the ribosome itself. Furthermore, we isolated decapped mRNA degradation intermediates from ribosome complexes and performed high-throughput sequencing analysis. Interestingly, 93% of the decapped mRNA fragments (approximately 12,000) could be detected at the same relative abundance on ribosome complexes and in cell lysates. In summary, our findings strongly indicate the association of the majority of bulk mRNAs as well as mRNAs targeted by miRNAs with the ribosome during their degradation.     

Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3'' noncoding region is mediated by a cotranslational mechanism.

The 3' noncoding region element (AUUUA)n specifically targets many short-lived mRNAs for degradation. Although the mechanism by which this sequence functions is not yet understood, a potential link between facilitated mRNA turnover and translation has been implied by the stabilization of cellular mRNAs in the presence of protein synthesis inhibitors. We therefore directly investigated the role of translation on mRNA stability. We demonstrate that mRNAs which are poorly translated through the introduction of stable secondary structure in the 5' noncoding region are not efficiently targeted for selective destabilization by the (AUUUA)n element. These results suggest that AUUUA-mediated degradation involves either a 5'-->3' exonuclease or is coupled to ongoing translation of the mRNA. To distinguish between these two possibilities, we inserted the poliovirus internal ribosome entry site, which promotes internal ribosome initiation, downstream of the 5' secondary structure. Translation directed by internal ribosome binding was found to fully restore targeted destabilization of AUUUA-containing mRNAs despite the presence of 5' secondary structure. This study therefore demonstrates that selective degradation mediated by the (AUUUA)n element is coupled to ribosome binding or ongoing translation of the mRNA and does not involve 5'-to-3' exonuclease activity.