Robust Pixel Design Methodologies for a Vertical Avalanche Photodiode (VAPD)-Based CMOS Image Sensor.

We present robust pixel design methodologies for a vertical avalanche photodiode-based CMOS image sensor, taking account of three critical practical factors: (i) "guard-ring-free" pixel isolation layout, (ii) device characteristics "insensitive" to applied voltage and temperature, and (iii) stable operation subject to intense light exposure. The "guard-ring-free" pixel design is established by resolving the tradeoff relationship between electric field concentration and pixel isolation. The effectiveness of the optimization strategy is validated both by simulation and experiment. To realize insensitivity to voltage and temperature variations, a global feedback resistor is shown to effectively suppress variations in device characteristics such as photon detection efficiency and dark count rate. An in-pixel overflow transistor is also introduced to enhance the resistance to strong illumination. The robustness of the fabricated VAPD-CIS is verified by characterization of 122 different chips and through a high-temperature and intense-light-illumination operation test with 5 chips, conducted at 125 °C for 1000 h subject to 940 nm light exposure equivalent to 10 kLux.

The cap-specific m6A methyltransferase, PCIF1/CAPAM, is dynamically recruited to the gene promoter in a transcription-dependent manner.

N 6-methyladenosine (m6A), the most abundant modification in eukaryotic mRNAs, plays an important role in mRNA metabolism and functions. When adenosine is transcribed as the first cap-adjacent nucleotide, it is methylated at the ribose 2'-O and N6 positions, thus generating N6, 2'-O-dimethyladenosine (m6Am). Phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1) is a novel cap-specific adenine N6-methyltransferase responsible for m6Am formation. As PCIF1 specifically interacts with the Ser5-phosphorylated CTD of RNA polymerase II (Pol II), which is a marker for the early phase of transcription, PCIF1 is speculated to be recruited to the early elongating Pol II. In this study, subcellular fractionation and immunofluorescence microscopy demonstrated that PCIF1 is mainly localized to the transcriptionally active chromatin regions in HeLa cells. Chromatin immunoprecipitation (ChIP) revealed that PCIF1 was predominantly localized to the promoter of a broad range of Pol II-transcribed genes, including several protein-coding genes and non-coding RNA genes. Moreover, PCIF1 accumulation on these promoters depended entirely on transcriptional activity and Ser5 phosphorylation of the CTD. These results suggest that PCIF1 dynamically localizes to the Pol II early in transcription and may efficiently catalyze N6-methylation of the first adenosine residue of nascent mRNAs cotranscriptionally.

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants.

PURPOSE: To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. METHODS: Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19G28R and CDK19Y32H. RESULTS: We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). CONCLUSION: CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.

Modeling and Analysis of Capacitive Relaxation Quenching in a Single Photon Avalanche Diode (SPAD) Applied to a CMOS Image Sensor.

We present an analysis of carrier dynamics of the single-photon detection process, i.e., from Geiger mode pulse generation to its quenching, in a single-photon avalanche diode (SPAD). The device is modeled by a parallel circuit of a SPAD and a capacitance representing both space charge accumulation inside the SPAD and parasitic components. The carrier dynamics inside the SPAD is described by time-dependent bipolar-coupled continuity equations (BCE). Numerical solutions of BCE show that the entire process completes within a few hundreds of picoseconds. More importantly, we find that the total amount of charges stored on the series capacitance gives rise to a voltage swing of the internal bias of SPAD twice of the excess bias voltage with respect to the breakdown voltage. This, in turn, gives a design methodology to control precisely generated charges and enables one to use SPADs as conventional photodiodes (PDs) in a four transistor pixel of a complementary metal-oxide-semiconductor (CMOS) image sensor (CIS) with short exposure time and without carrier overflow. Such operation is demonstrated by experiments with a 6 µm size 400 × 400 pixels SPAD-based CIS designed with this methodology.

Cap-specific terminal N 6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

N 6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N 6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N 6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N 6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

A 250 m Direct Time-of-Flight Ranging System Based on a Synthesis of Sub-Ranging Images and a Vertical Avalanche Photo-Diodes (VAPD) CMOS Image Sensor.

We have developed a direct time-of-flight (TOF) 250 m ranging Complementary Metal Oxide Semiconductor (CMOS) image sensor (CIS) based on a 688 × 384 pixels array of vertical avalanche photodiodes (VAPD). Each pixel of the CIS comprises VAPD with a standard four transistor pixel circuit equipped with an analogue capacitor to accumulate or count avalanche pulses. High power near infrared (NIR) short (<50 ns) and repetitive (6 kHz) laser pulses are illuminated through a diffusing optics. By globally gating the VAPD, each pulse is counted in the in-pixel counter enabling extraction of sub-photon level signal. Depth map imaging with a 10 cm lateral resolution is realized from 1 m to 250 m range by synthesizing subranges images of photon counts. Advantages and limitation of an in-pixel circuit are described. The developed CIS is expected to supersede insufficient resolution of the conventional light detection and ranging (LiDAR) systems and the short range of indirect CIS TOF.

A Real-Time Ultraviolet Radiation Imaging System Using an Organic Photoconductive Image Sensor.

We have developed a real time ultraviolet (UV) imaging system that can visualize both invisible UV light and a visible (VIS) background scene in an outdoor environment. As a UV/VIS image sensor, an organic photoconductive film (OPF) imager is employed. The OPF has an intrinsically higher sensitivity in the UV wavelength region than those of conventional consumer Complementary Metal Oxide Semiconductor (CMOS) image sensors (CIS) or Charge Coupled Devices (CCD). As particular examples, imaging of hydrogen flame and of corona discharge is demonstrated. UV images overlapped on background scenes are simply made by on-board background subtraction. The system is capable of imaging weaker UV signals by four orders of magnitude than that of VIS background. It is applicable not only to future hydrogen supply stations but also to other UV/VIS monitor systems requiring UV sensitivity under strong visible radiation environment such as power supply substations.

Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance.

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

Mediator cyclin-dependent kinases upregulate transcription of inflammatory genes in cooperation with NF-κB and C/EBPß on stimulation of Toll-like receptor 9.

In eukaryotes, the Mediator complex has important roles in regulation of transcription by RNA polymerase II. Mediator is a large complex with more than 20 subunits that form head, middle, tail and CDK/cyclin modules. Among them, CDK8 and/or CDK19 (CDK8/19), and their counterpart cyclin C, form the CDK/cyclin module together with Mediator subunits MED12 and MED13. Despite evidences of both activation and repression, the precise functional roles of CDK8/19 in transcription are still elusive. Our previous results indicate that CDK8/19 recruits epigenetic regulators to repress immunoresponse genes. Here, this study focused on Toll-like receptors (TLRs), which exert innate immune responses through recognition of pathogen-associated molecular patterns and examined the functional roles of CDK8/19. As a result, CDK8/19 regulated transcription of inflammatory genes on stimulation of TLR9 in myeloma-derived RPMI8226 cells, which led to expression of inflammation-associated genes such as IL8, IL10, PTX3 and CCL2. Mediator subunits CDK8/19 and MED1, inflammation-related transcriptional activator NF-κB and C/EBPß, and general transcription factors TFIIE and TFIIB colocalized at the promoter regions of these genes under this condition. Our results show that CDK8/19 positively regulates inflammatory gene transcription in cooperation with NF-κB and C/EBPß on stimulation of TLR9.

Human SCP4 is a chromatin-associated CTD phosphatase and exhibits the dynamic translocation during erythroid differentiation.

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit contains tandem repeats of the heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is subject to dynamic phosphorylation during transcription, mainly at serine residues (Ser2, Ser5 and Ser7). Regulation of CTD phosphorylation by specific kinases and phosphatases is crucial for coordinating transcription with RNA processing and histone modification. Human small CTD phosphatase 4 (SCP4), also called CTDSPL2 or HSPC129, is a putative CTD phosphatase belonging to the FCP/SCP family and implicated in control of ε- and γ-globin gene expression. Here, we report the biochemical and functional characterization of SCP4. SCP4 exhibited Ser5-preferential CTD phosphatase activity in vitro, while small interfering RNA-mediated SCP4 knockdown in HeLa cells increased phosphorylation levels of Pol II at Ser5 and Ser7, but not at Ser2. Furthermore, cell fractionation, chromatin immunoprecipitation and immunofluorescence assays revealed an exclusive localization for SCP4 in the chromatin, particularly at transcriptionally silenced chromosomal regions. Interestingly, SCP4 was gradually released from the chromatin fraction during hemin-induced erythroid differentiation of K562 cells, with concomitant cytoplasmic accumulation. Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation.

Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation.

Human mediator MED17 subunit plays essential roles in gene regulation by associating with the transcription and DNA repair machineries.

In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.

Transcription cofactor PC4 plays essential roles in collaboration with the small subunit of general transcription factor TFIIE.

In eukaryotes, positive cofactor 4 (PC4) stimulates activator-dependent transcription by facilitating transcription initiation and the transition from initiation to elongation. It also forms homodimers and binds to single-stranded DNA and various transcriptional activators, including the general transcription factor TFIIH. In this study, we further investigated PC4 from Homo sapiens and the nematode Caenorhabditis elegans (hPC4 and cePC4, respectively). hPC4 strongly stimulated transcription on a linearized template, whereas it alleviated transcription on a supercoiled template. Transcriptional stimulation by PC4 was also alleviated by increasing the amount of TFIID. GST pull-down studies with general transcription factors indicated that both hPC4 and cePC4 bind strongly to TFIIB, TFIIEß, TFIIFα, TFIIFß and TFIIH XPB subunits and weakly to TBP and TFIIH p62. However, only hPC4 bound to CDK7. The effect of each PC4 on transcription was studied in combination with TFIIEß. hPC4 stimulated both basal and activated transcription, whereas cePC4 primarily stimulated activated transcription, especially in the presence of TFIIEß from C. elegans. Finally, hPC4 bound to the C-terminal region of hTFIIEß adjacent to the basic region. These results indicate that PC4 plays essential roles in the transition step from transcription initiation to elongation by binding to melted DNA in collaboration with TFIIEß.

Vertebrate Ssu72 regulates and coordinates 3'-end formation of RNAs transcribed by RNA polymerase II.

In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3'-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity in vitro, but its roles in gene expression and CTD dephosphorylation in vivo remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3'-end formation of U2 and U4 snRNAs and GAPDH mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3'-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3'-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3'-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3'-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.

Human RNA polymerase II-associated protein 2 (RPAP2) interacts directly with the RNA polymerase II subunit Rpb6 and participates in pre-mRNA 3'-end formation.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD of Pol II undergoes reversible phosphorylation during the transcription cycle, mainly at Ser2, Ser5, and Ser7. Dynamic changes in the phosphorylation patterns of the CTD are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Human RNA polymerase II-associated protein 2 (RPAP2) was originally identified as a Pol II-associated protein and was subsequently shown to function as a novel Ser5-specific CTD phosphatase. Although a recent study suggested that RPAP2 is required for the efficient expression of small nuclear RNA genes, the role of RPAP2 in controlling the expression of protein-coding genes is unknown. Here, we demonstrate that the C-terminal region of RPAP2 interacts directly with the Pol II subunit Rpb6. Chromatin immunoprecipitation analyses of the MYC and GAPDH protein-coding genes revealed that RPAP2 occupied the coding and 3' regions. Notably, siRNA-mediated knockdown of RPAP2 caused defects in 3'-end formation of the MYC and GAPDH pre-mRNAs. These results suggest that RPAP2 controls Pol II activity through a direct interaction with Rpb6 and participates in pre-mRNA 3'-end formation.

Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes.

A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization.

Mediator CDK subunits are platforms for interactions with various chromatin regulatory complexes.

The Mediator complex consists of more than 20 subunits. This is composed of four modules: head, middle, tail and CDK/Cyclin. Importantly, Mediator complex is known to play pivotal roles in transcriptional regulation, but its molecular mechanisms are still elusive. Many studies, including our own, have revealed that CDK8, a kinase subunit of the CDK/Cyclin module, is one of the key subunits involved in these roles. Additionally, we previously demonstrated that a novel CDK component, CDK19, played similar roles. It is assumed that various factors that directly affect transcriptional regulation target these two CDKs; thus, we conducted yeast two-hybrid screenings to isolate the CDK19-interacting proteins. From a screening of 40 million colonies, we obtained 287 clones that provided positive results encoded mRNAs, and it turned out that 59 clones of them encoded nuclear proteins. We checked the reading frames of the candidate clones and obtained three positive clones, all of which encoded the transcriptional cofactors, Brahma-related gene 1, B-cell CLL/lymphoma 6 and suppressor of zeste 12 homolog. Intriguingly, these three cofactors are also related to chromatin regulation. Further studies demonstrated that those could bind not only to CDK19 but also to CDK8. These results help elucidate the functional mechanism for the mutual regulations between transcription and chromatin.

Identification of target genes for the CDK subunits of the Mediator complex.

Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate the in vivo transcriptional activities of the complexes, we used a luciferase assay in combined with siRNA-mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16-dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context-specific transcriptional regulator.

The HIRA complex subunit Hip3 plays important roles in the silencing of meiosis-specific genes in Schizosaccharomyces pombe.

BACKGROUND: The control of gene expression is essential for growth and responses to environmental changes in various organisms. It is known that some meiosis-specific genes are silenced during mitosis and expressed upon nitrogen starvation in Schizosaccharomyces pombe. When the factors responsible for this regulation were studied, a hip3 mutant was isolated via discovery of a defect in the transcriptional repression of meiosis-specific genes. Hip3 is a subunit of the HIRA (histone regulatory complex A) complex, which consists of four subunits (Hip1, Hip3, Hip4 and Slm9) and acts as a histone chaperone that is independent of DNA replication. METHODOLOGY/PRINCIPAL FINDINGS: In a search for mutants, the meiosis-specific gene SPCC663.14c(+) was identified by screening for genes that are silenced during mitosis and induced upon nitrogen starvation. A reporter plasmid that expresses the ura4(+) gene driven by the SPCC663.14c(+) promoter was constructed. Screening for suppressor mutants was then carried out in nitrogen-rich medium without uracil. A mutant with a mutation in the hip3(+) gene was isolated and named hip3-1. This mutation alleviated the transcriptional repression of the ura4(+) gene on the reporter plasmid and of the endogenous SPCC663.14c(+) gene in the presence of nitrogen. A ChIP assay revealed that RNA polymerase II (Pol II) and TFIIE were enriched at the SPCC663.14c(+) locus, whereas the levels of histone H3 were decreased in hip3-1 cells. Intriguingly, histone H3 was heavily modified at the SPCC663.14c(+) locus in hip3-1 cells; these modifications included tri-methylation and acetylation of H3 lysine 9 (H3K9), mono-methylation of H3 arginine 2 (H3R2), and tri-methylation of H3 lysine 4 (H3K4). In addition, the tri-methylation of H3K9 and H3K4 were strongly elevated in hip3-1 mutants. CONCLUSIONS: Taken together, these results indicate that Hip3 plays important roles in the control of histone modifications at meiosis-specific gene loci and induces their transcriptional repression.

Enhancing recombinant protein production in human cell lines with a constitutive transport element and mRNA export proteins.

Recent research into mRNA maturation processes in the nucleus has identified a number of proteins involved in mRNA transcription, capping, splicing, end processing and export. Among them, the Tap-p15 heterodimer acts as an mRNA export receptor. Tap-p15 is recruited onto fully processed mRNA in the nucleus, which is ready for export to the cytoplasm, through associating with Aly or SR proteins on mRNA, or by directly associating with a constitutive transport element (CTE), an RNA element derived from type D retroviruses. mRNA containing a CTE is exported to the cytoplasm by directly associating with Tap-p15, even in the absence of Tap-recruiting proteins such as Aly or SR proteins on the mRNA. Here, we showed that the use of a CTE enhanced the expression of recombinant protein in human cell lines. The co-expression of reporter proteins and Tap-p15 also enhanced recombinant protein expression. Moreover, the use of a CTE and Tap-p15 synergistically further enhanced the recombinant protein expression. In addition to Tap-p15, several Tap-p15-recruiting proteins, including Aly and SR proteins, enhanced recombinant protein expression, albeit independently of the CTE. The incorporation of a CTE and Tap-p15-recruiting proteins into protein expression system is useful to increase recombinant protein yield in human cells.