Improving translation initiation site and stop codon recognition by using more than two classes

Motivation: The recognition of translation initiation sites and stop codons is a fundamental part of any gene recognition program. Currently, the most successful methods use powerful classifiers, such as support vector machines with various string kernels. These methods all use two classes, one of positive instances and another one of negative instances that are constructed using sequences from the whole genome. However, the features of the negative sequences differ depending on the position of the negative samples in the gene. There are differences depending on whether they are from exons, introns, intergenic regions or any other functional part of the genome. Thus, the positive class is fairly homogeneous, as all its sequences come from the same part of the gene, but the negative class is composed of different instances. The classifier suffers from this problem. In this article, we propose the training of different classifiers with different negative, more homogeneous, classes and the combination of these classifiers for improved accuracy. Results: The proposed method achieves better accuracy than the best state-of-the-art method, both in terms of the geometric mean of the specificity and sensitivity and the area under the receiver operating characteristic and precision recall curves. The method is tested on the whole human genome. The results for recognizing both translation initiation sites and stop codons indicated improvements in the rates of both false-negative results (FN) and false-positive results (FP). On an average, for translation initiation site recognition, the false-negative ratio was reduced by 30.2% and the FP ratio decreased by 10.9%. For stop codon prediction, FP were reduced by 41.4% and FN by 31.7%. Availability and implementation: The source code is licensed under the General Public License and is thus freely available. The datasets and source code can be obtained from http://cib.uco.es/site-recognition. Contact: [email protected]

Selection and characterization of RNA aptamers that detect a quaternary structure for ribosomal protein S7

Here we report on the selection and characterization of RNA aptamers that recognize E. coli ribosomal protein S7. Ribosomal protein S7 plays two important roles in ribosome biogenesis: (1) as an assembly initiator, S7 nucleates the folding of the 3\u27 major domain of 16S rRNA, and (2) it binds to the str operon and represses the translation of S12, S7, and EF-G. The primary and secondary structures of the S7 binding sites of rRNA and mRNA share limited sequence and structural homology and the required elements for high affinity binding have not been entirely elucidated. We have selected RNA aptamers that share very little primary sequence homology to either the S7 binding site of 16S rRNA or to the intercistronic region of str mRNA. Many of the aptamers are expected to fold into three-helix junctions, a structure particularly reminiscent of the mRNA. Interestingly, the aptamers exhibit cooperative binding with Hill coefficients of ~3 indicating that they are detecting a quaternary structure of S7. We have found that the S7 aptamers use the same amino acids and structural elements to bind S7 as the rRNA and mRNA indicating that the same binding site is used for all three RNAs. With gel filtration, we were only able to isolate the aptamer/S7 complex at a 1:1 stoichiometry, indicating that the proposed quaternary structure of S7 is weak. However, deletion of the β-ribbon nearly eliminates cooperative aptamer binding suggesting that this structural element may be involved in protein-protein interaction. Furthermore, pre-treatment of native S7 with the N-terminal extension also results in a significant reduction in cooperative aptamer binding. The results presented here suggest that S7 itself may undergo conformational rearrangement subsequent to 16S rRNA binding, and may help explain the strong temperature-dependent rearrangements at the binding site of S7 within the 16S rRNA. Furthermore, we propose that the weak, multimeric interaction of S7 may have a role in the retroregulation of S12. S7 may bind to the mRNA in a pre-multimerized form or multimerize subsequent to binding, resulting in ribosome stalling due to the multimeric obstacle. If the S7/S7 interaction is weak however, then it may be easily disrupted by repeated ribosome bombardment, causing eventual decay of the multimer and relieving some of the translational repression. Translational repression of the genes encoding S7 and EF-G would remain constant over time however, because the monomeric S7 bound more tightly to the intercistronic region would continue to prevent translational coupling with the upstream gene encoding S12

Mechanistic Insights into Translational Silencing of HACL mRNA in Yeast S. Cerevisiae

Protein synthesis is a fundamental life process. Protein synthesis regulates cellular metabolism, cellular growth, the cell cycle, and cell morphogenesis. Technical advances in molecular biology, advanced high-throughput sequencing technologies and recent developments in crystallographic methodologies have aided in better understanding of the process of protein synthesis. Current knowledge of protein synthesis provides us with an overview of the initiation, elongation, and termination steps, general regulatory mechanisms, and molecular functions of the ribosomal rRNA and proteins. However, even with all this information we are far from understanding the detailed sequence of molecular interactions involved in the process of protein synthesis. We have snapshots of different stages of protein synthesis (initiation, elongation, and termination) but, we are still missing the intricate details responsible for linking these snapshots and creating a complete picture depicting the mechanism of protein synthesis. This thesis is focused on understanding the mechanism of protein synthesis using the yeast HAC1 mRNA as a model mRNA in yeast Saccharomyces cerevisiae. The messenger RNA (mRNA) bears the genetic information that is decoded in the process of protein synthesis. The fate of a messenger mRNA is decided by regulatory elements present in the mRNA that are known as cis-acting elements. They influence not only translation of the mRNA, but also mRNA splicing, mRNA localization, mRNA processing, and mRNA degradation. One of the cis-regulating factors is the RNA secondary structure. The RNA secondary structure controls the translation of an mRNA. The precise mechanism of which is not clearly understood. This thesis is aimed at uncovering the regulation of protein synthesis by the RNA secondary structure. Proteins in their native conformation either spontaneously attain the folded conformation or they are folded by chaperone proteins in the cytoplasm or in the endoplasmic reticulum (ER). Sometimes the ER experiences an overload of unfolded proteins which results in a condition termed as “ER stress”. Under the stress conditions, unfolded protein response (UPR) pathways are activated which result in expression of assorted transcription factors. These transcription factors modulate the cellular transcriptome and proteome to alleviate the ER stress conditions. Our model mRNA from Saccharomyces cerevisiae encodes a transcription factor that is expressed under conditions of ER stress. HAC1 mRNA contains a cytoplasmic intron (252 nucleotides) that base-pairs with the 5’- untranslated region (5’-UTR) of the mRNA. This base-pairing interaction inhibits the translation of the HAC1 mRNA under physiological conditions. We use HAC1 mRNA and the inherent base-pairing interaction to uncover new insights into the mechanism of translational regulation by the RNA secondary structure. HAC1 pre-mRNA is composed of a 5’-untranslated region {(5’-UTR, 68-nucleotides (nt)}, an exon1 (661-nt), an intron (252-nt), an exon2 (57-nt), and a 3’-UTR (462-nt). Under conditions of ER stress, an endonuclease Ire1 cleaves the intron from the HAC1 pre-mRNA to relieve the translational block of the mRNA. The two exons are spliced by tRNA ligase and the mature HAC1 mRNA produces Hac1 protein which is an active transcription factor. We have shown that the base pairing interaction between 5’-UTR and intron inhibits translation initiation of HAC1 mRNA [1]. We performed a random genetic screen to identify an intragenic suppressor mutation (s) that would overcome the translational block in HAC1 mRNA. We identified a point mutation in the base-pairing interaction that relieved the translational block in HAC1 mRNA. Further mutational analyses of the base-pairing interaction suggested that it is critical for the regulation of HAC1 mRNA translation. We also showed that insertion of an in-frame AUG start codon upstream of the RNA secondary structure releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. Together, we showed that the RNA secondary structure regulates translation initiation of HAC1 mRNA [1]. To dissect the translation initiation block further we shifted the RNA secondary structure from its normal position (which is cap-proximal) to one away from the mRNA cap structure (a cap-distal position) by inserting an unstructured RNA sequence. We observed that genetically engineered HAC1 mRNA with the cap-distal secondary structure resulted in translation of the Hac1 protein. This result suggested that the cap-proximal RNA secondary structure possibly inhibits the interaction of the pre-initiation complex (PIC) with the HAC1 mRNA. Further analyses of the yeast transcriptome and translatome suggest that the HAC1 gene locus expresses two overlapping transcripts; referred to here as “HAC1a” like the one described above, and “HAC1b”, a newly identified variant. Interestingly, the newly identified HAC1b mRNA overlaps with the exon1 of HAC1a. Yeast transcriptome analyses show that it is composed of a long 5’-UTR (~400-nt), an open reading frame (693-nt) and a short 3’-UTR (124-nt). We characterized the role of the HAC1b transcript in the context of the ER stress response. We observed that HAC1b, like HAC1a mRNA, remains translationally silent. However, HAC1b can activate the ER stress response as a functional Hac1 protein is synthesized in the absence of “Duh1” protein. “Duh1” is a component of the proteasome complex. These observations are consistent with the previous report that Duh1 targets the protein product from the un-spliced HAC1 mRNA for degradation. Taken together, our results provide mechanistic insights into the translational regulation of HAC1 mRNA. In addition, we provide evidence that the transcript isoform of HAC1 mRNA might play a role in the ER stress response in yeast, S. cerevisiae

The influence of microRNAs and poly(A) tail length on endogenous mRNA–protein complexes

Background: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. Results: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. Conclusions: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.National Institutes of Health (U.S.) (Grant K99GM102319)National Institutes of Health (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Grant R01GM067031)National Institutes of Health (U.S.) (Grant R35GM118135)Natural Sciences and Engineering Research Council of Canada (Discovery Grant

Untranslated yet indispensable - UTRs act as key regulators in the environmental control of gene expression

To survive and thrive in a dynamic environment, plants must continuously monitor their surroundings and adjust their development and physiology accordingly. Changes in gene expression underlie these developmental and physiological adjustments and are traditionally attributed to widespread transcriptional reprogramming. Growing evidence, however, suggests that post-transcriptional mechanisms also play a vital role in tailoring gene expression to a plant's environment. Untranslated regions (UTRs) act as regulatory hubs for post-transcriptional control, harbouring cis elements that affect an mRNA's processing, localisation, translation and stability and thereby tune the abundance of the encoded protein. Here, we review recent advances made in understanding the critical function UTRs exert in the post-transcriptional control of gene expression in the context of a plant's abiotic environment. We summarise the molecular mechanisms at play, present examples of UTR-controlled signalling cascades and discuss the potential that resides within UTRs to render plants more resilient to a changing climate.</p

Structural basis of translational recycling and bacterial ribosome rescue

In the last step of gene expression, a messenger RNA (mRNA) sequence is translated into a polypeptide. This highly regulated and dynamic process is carried out by the ribosome, a ribonucleoprotein complex composed of two unequal subunits. The translation cycle is initiated when the small ribosomal subunit (SSU) binds to an mRNA and recognizes the start codon of the open reading frame (ORF). Then the large ribosomal subunit (LSU) joins and the ribosome starts moving along the mRNA. A protein is synthesized until the ribosome reaches a stop codon. A cell needs thousands (prokaryotes) or millions (eukaryotes) of ribosomes for protein production and spends enormous amounts of energy on the assembly of this macromolecular machinery. Therefore, it is crucial to recycle the machinery after each successful round of translation. The recycling step allows release of mRNA, transfer RNA (tRNA) and the synthesized polypeptide from ribosomal subunits and subsequent binding of the next mRNA for protein synthesis. The first part of this dissertation includes studies of the highly conserved and essential ribosome recycling factor ATP binding cassette (ABC) Subfamily E Member 1 (ABCE1). In eukaryotes and archaea, ABCE1 binds the ribosome and in concert with an A-site factor and splits the ribosome into large and small subunits. ABCE1 harbors two nucleotide binding sites (NBSs), which are formed at the interface of two nucleotide binding domains (NBDs). Prior to this work, the ABCE1-bound pre-splitting complex, as well as the ABCE1-bound post-splitting complex, had been visualized by cryo-electron microscopy (cryo-EM) at medium resolution. This structural analysis combined with functional studies led to a model for the mechanism of the splitting event. ATP-binding and the closure of the NBSs lead to repositioning of the iron-sulfur cluster domain, which results in collision with the A-site factor and ribosome splitting. Yet, how conformational changes during the splitting event are triggered and communicated to the NBSs of ABCE1, was not understood. To gain molecular insights into this process, a structure of a fully nucleotide-occluded (closed) state of ABCE1 bound to the archaeal 30S post-splitting complex was solved by cryo-EM. At a resolution of 2.8 Å a detailed molecular analysis of ABCE1 was performed and confirmed by a combination of mutational and functional studies. This allowed to propose a refined model of how the ATPase cycle is linked to ribosome splitting and which role the different domains of ABCE1 play. In eukaryotes, the recycling phase is directly linked to translation initiation via the SSU. After being released from the mRNA 3’ end, the SSU can engage with another or even the same mRNA at the 5’ end. The recycling factor ABCE1 was found to be associated with initiation complexes, but whether it plays a role in initiation was not clear. Using cryo-EM, structures of native ABCE1-containing initiation complexes were solved and intensive 3D classification allowed to distinguish different stages of initiation, during which ABCE1 may play a role. Surprisingly, ABCE1 adopted a previously unknown state for ABC-type ATPases that was termed “hybrid state”. Here, the NBSI is in a half open state with ADP bound and the NBSII is in a closed state with ATP bound. Further, eukaryotic initiation factor 3j (eIF3j) was found to stabilize this hybrid conformation via its N-terminus. Since eIF3j had already been described to assist ABCE1 in ribosome dissociation, in vitro splitting assays were performed demonstrating that eiF3j indeed actively enhances the splitting reaction. On top of this, the high-resolution structure allowed to describe the interaction network of eIF3j with the ribosome, initiation factors (IFs), and ABCE1. Independent of ABCE1, the structures presented here allowed to provide an improved molecular model of the human 43S pre-initiation complex (PIC) and to analyze its sophisticated interaction network. In particular, new molecular insights into the large eIF3 complex encircling the 43S PIC, and the eIF2 ternary complex delivering the initiator tRNA are provided. Equally important as canonical recycling is the recognition and recycling of ribosomes that result from translational failure. Aberrant translation elongation and ribosome stalling can be caused by a plethora of different stresses. In bacterial cells, multiple rescue systems are known such as trans-translation or alternative ribosome rescue factor-mediated termination, which act on ribosome nascent chain complexes with an empty A-site (non-stop complexes). It has been a long standing question how ribosomes that are stalled in the middle of an ORF (no-go complexes) are recognized and recycled. The second part of this dissertation reports a new bacterial rescue system that acts on no-go complexes. In eukaryotes, the concept of ribosome collisions as a trigger for ribosome rescue has been studied extensively. Here, it was found that a similar mechanism exists in bacteria and thus a structural analysis of collided disomes in E. coli and B. subtilis was conducted. In a genetic screen, the endonuclease SmrB was identified as one candidate for a collision sensor. Structural analysis of SmrB-bound disomes elucidated how this rescue factor is recruited to collided ribosomes. Its SMR domain binds to the disome interface between the stalled and the collided ribosome in close proximity to the mRNA and in a position ideal to perform endonucleolytic cleavage. Such cleavage then results in non-stop complexes that can be recycled by the pathways mentioned above. In conclusion, this work provides mechanistic insights into how a cell distinguishes stalled ribosomes from actively translating ribosomes and characterizes a novel ribosome rescue pathway

Characterization of endogenous Kv1.3 channel isoforms in T cells.

The voltage-gated potassium channel Kv1.3 plays a crucial role in T-cell activation and is considered a promising target for the treatment of autoimmune diseases. However, the lack of reliable antibodies has prevented its accurate detection and study under endogenous conditions, so that most published studies have been conducted in heterologous systems. To address this limitation, we engineered a Jurkat T-cell line expressing endogenous Kv1.3 channels tagged with a signal peptide to investigate the expression and localization of native Kv1.3 channels, and their role associated to T cell physiological responses. Using the CRISPR-Cas9 tool, we inserted a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal and activated channel expression were assessed through western blot analysis and imaging techniques. Surprisingly, besides the canonical Kv1.3 channel (54 KDa), we identified two additional isoforms with distinct N termini: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms showed upregulation after T-cell activation. Our focus was on characterizing the truncated isoform (short form, SF) as it had not been previously described and could be present in available Kv1.3-/- mouse models. Overexpressing SF in HEK cells generated Kv1.3-like currents with smaller amplitudes, which, unlike canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. While the canonical isoform localized primarily at the plasma membrane, SF remained intracellular, accumulating perinuclearly. Consequently, SF Jurkat cells lacked Kv1.3 currents, exhibited depolarized resting membrane potential (EM), reduced Ca2+ influx, and diminished increases in intracellular calcium ([Ca2+]i) upon stimulation. Functional characterization of these Kv1.3 channel isoforms revealed their differential contributions to signaling pathways involved in immunological synapse formation. In conclusion, alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells with distinct functional roles. Importantly, some of these functions do not require the formation of functional plasma membrane channels by Kv1.3 proteins.El canal de potasio dependiente de voltaje Kv1.3 juega un papel crucial en la activación de las células T y se considera una buena diana terapéutica para el tratamiento de enfermedades autoinmunes. Sin embargo, la falta de anticuerpos específicos de la proteína ha impedido su detección y estudio precisos en condiciones endógenas, por lo que la mayoría de los estudios publicados se han realizado en sistemas heterólogos. Para abordar esta limitación, diseñamos una línea de células T Jurkat que expresa canales Kv1.3 endógenos marcados con un péptido señal para estudiar su expresión y localización además de su papel fisiológico en los linfocitos T. Usando la herramienta CRISPR-Cas9, insertamos un péptido Flag-Myc en el extremo C del gen KCNA3. La expresión del canal basal y activado se evaluó mediante análisis de transferencia Western y técnicas de imagen. Sorprendentemente, además del canal canónico Kv1.3 (54 KDa), identificamos dos isoformas adicionales con extremos N distintos: una isoforma más larga (70 KDa) y una isoforma truncada (43 KDa). Las tres isoformas mostraron un aumento de su expresión después de la activación de las células T. Nuestro objetivo fue caracterizar la isoforma truncada (forma abreviada, SF) ya que no se había descrito previamente y podría estar presente en los modelos de ratón Kv1.3-/- disponibles. La sobreexpresión de SF en células HEK generó corrientes similares a Kv1.3 con amplitudes más pequeñas que, a diferencia del Kv1.3 canónico, no indujeron la proliferación de HEK. Para explorar el papel de la isoforma SF endógena en un sistema nativo, generamos un clon de Jurkat knockout y un clon que expresa solo la isoforma SF. Mientras que la isoforma canónica se localizó principalmente en la membrana plasmática, el SF permaneció intracelular, acumulándose perinuclearmente. En consecuencia, las células SF Jurkat carecían de las corrientes Kv1.3, presentaban un potencial de membrana en reposo (EM) despolarizado, una entrada de Ca2+ reducida y un aumento disminuido del calcio intracelular ([Ca2+]i) tras la estimulación. La caracterización funcional de estas isoformas del canal Kv1.3 reveló sus contribuciones diferenciales a las vías de señalización involucradas en la formación de sinapsis inmunológicas. En conclusión, el inicio de la traducción alternativa genera al menos tres isoformas endógenas del canal Kv1.3 en las células T con distintas funciones funcionales. Es importante destacar que algunas de estas funciones no requieren la formación de canales de membrana plasmática funcionales por las proteínas Kv1.3.Escuela de DoctoradoDoctorado en Investigación Biomédic

What Controls the Controller: Structure and Function Characterizations of Transcription Factor PU.1 Uncover Its Regulatory Mechanism

The ETS family transcription factor PU.1/Spi-1 is a master regulator of the self-renewal of hematopoietic stem cells and their differentiation along both major lymphoid and myeloid branches. PU.1 activity is determined in a dosage-dependent manner as a function of both its expression and real-time regulation at the DNA level. While control of PU.1 expression is well established, the molecular mechanisms of its real-time regulation remain elusive. Our work is focused on discovering a complete regulatory mechanism that governs the molecular interactions of PU.1. Structurally, PU.1 exhibits a classic transcription factor architecture in which intrinsically disordered regions (IDR), consisting of 66% of its primary structure, are tethered to a well-structured DNA binding domain. The transcriptionally active form of PU.1 is a monomer that binds target DNA sites as a 1:1 complex. Our investigations show that IDRs of PU.1 reciprocally control two separate inactive dimeric forms, with and without DNA. At high concentrations, PU.1 forms a non-canonical 2:1 complex at a single DNA specific site. In the absence of DNA, PU.1 also forms a dimer, but it is incompatible with DNA binding. The DNA-free PU.1 dimer is further promoted by phosphomimetic mutants of IDR residues that are phosphorylated in B-lymphocytic activation. These results lead us to postulate a model of real-time PU.1 regulation, unknown in the ETS family, where independent dimeric forms antagonize each other to control the dosage of active PU.1 monomer at its target DNA sites. To demonstrate the biological relevance of our model, cellular assays probing PU.1-specific reporters and native target genes show that PU.1 transactivation exhibits a distinct dose response consistent with negative feedback. In summary, we have established the first model for the general real-time regulation of PU.1 at the DNA/protein level, without the need for recruiting specific binding partners. These novel interactions present potential therapeutic targets for correcting de-regulated PU.1 dosage in hematologic disorders, including leukemia, lymphoma, and myeloma

Role of RGS2 in Cellular Stress

Stresses from the external environment can disrupt cellular processes and result in damaging effects, such as the misfolding of proteins, which have been linked to several diseases. Regulator of G protein signalling 2 (RGS2) is upregulated by several forms of stress and can inhibit protein synthesis, an established response to stress typically achieved via the phosphorylation of the initiation factor, eIF2, to conserve energy and resources. Under reduced translation, some factors are selectively expressed via alternative translation mechanisms and these factors consequently may promote apoptosis. The molecular mechanisms mediating such opposing responses to stress are not well understood. Here, we suggested that RGS2 may be an important regulatory component in the cellular stress response and we hypothesized that RGS2 contributes to the response of cells to stress through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrated that the expression of RGS2 decreased total protein levels and significantly increased levels of factors linked to stress-induced apoptosis such as ATF4 and CHOP. Interestingly, expression of the eIF2Bε-interacting domain of RGS2 (RGS2eb) alone resulted in a 20-fold increase in caspase 3 activation which was not seen with full-length RGS2. Furthermore, we showed that these effects are translationally regulated and independent of eIF2 phosphorylation. Thus, we present a novel mechanism in the regulation of stress response by RGS2. These results also suggest that RGS2 may be pro-apoptotic and may potentially be an important target in stress-related pathologies

Translational control by the multi-KH domain protein Scp160

The control of mRNA translation mediated by RNA-binding proteins (RBPs) is a key player in modulating gene expression. In S. cerevisiae, the multi-KH domain protein Scp160 associates with a large number of mRNAs and is present on membrane-bound and, to a lesser extent, cytosolic polysomes. Its binding site on the ribosome is close to the mRNA exit tunnel and in vicinity to Asc1, which constitutes a binding platform for signaling molecules. The present study focused on the closer characterization of the Scp160-ribosome interaction and on the suggested function of Scp160 in modulating the translation of specific target mRNAs. Using affinity purifications, the partial RNA-dependence of the Scp160-ribosome association was confirmed. In contrast to published results, ribosome association was found to be only slightly reduced but not abolished in the absence of Asc1 or the last two KH domains. Furthermore, the putative elongation regulator Stm1 was identified as a co-purifier of Scp160. In subcellular fractionation experiments, RNA-binding mutants of Scp160 were present in the ribosome-free cytosolic fraction and therefore partially deficient in ribosome association and/or mRNP formation. However, no physiological conditions were found that equally induce a shift of wildtype Scp160 towards the cytosolic fraction. Within the scope of a translational profiling approach, microarray analyses of RNA isolated from sucrose density gradient fractions were performed and led to the identification of a set of mRNAs that shift their position within the gradients upon Scp160 depletion, indicating changes in their translation rates. Consistent with the membrane localization of Scp160, transcripts encoding secreted proteins were significantly enriched. Using immunoprecipitation and subsequent quantitative real-time PCR (qRT-PCR), the interaction of Scp160 with a subgroup of the identified targets was confirmed and it was shown that their binding is dependent on the conserved GXXG motifs in the two C-terminal KH domains of Scp160. Furthermore, data were obtained indicating that Scp160 can act as a translational activator on some of its target mRNAs, probably on the level of translation elongation. Finally, first evidence was provided that the translational misregulation of specific target transcripts may be involved in the polyploidization that is a hallmark of Scp160-deprived cells. In summary, these data substantiate the assumption that Scp160 is involved in translational regulation of a specific, functionally related subset of mRNAs. This finding is in good accordance with the emerging view that RBPs co-regulate multiple transcripts in order to allow faster adaptation to environmental changes