UTR Processing Research Articles

Abstract 5628: Aberrant 3'UTR processing regulates a tumor suppressor network in AML

Abstract Differentiation blockade is a hallmark of acute myeloid leukemia (AML). While inactivating mutations in tumor suppressor genes (TSGs) have been well-studied, non-genetic mechanisms that impair TSGs to promote myeloid differentiation and influence drug response remain poorly understood. In this study, we performed genome-wide loss-of-function CRISPR/Cas9 screens to identify suppressors of myeloid differentiation. These screens converged on the zinc finger transcriptional repressor, ZBTB7A whose ablation restricted myeloid maturation and promoted a dysregulated inflammatory state to protect leukemic cells from undergoing differentiation. Moreover, genetic deletion of ZBTB7A or its paralog, ZBTB7B impeded leukemia differentiation and conferred resistance to differentiation-inducing agents. Mechanistically, we found that leukemia cells hijack 3’UTR processes to reduce mRNA expression of the tumor suppressor gene paralogs ZBTB7A and ZBTB7B. These findings provide a molecular framework of altered 3’UTR regulation that orchestrate a tumor suppressive function and promote resistance to AML differentiation-based therapies. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Sakthi Harini Rajendran, Sadik Karma, Nidhi Hariramani, Iannis Aifantis, Aristotelis Tsirigos. Aberrant 3'UTR processing regulates a tumor suppressor network in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5628.

PABPN1 aggregation is driven by Ala expansion and poly(A)-RNA binding, leading to CFIm25 sequestration that impairs alternative polyadenylation

Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.

Abstract 4741: NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML

Abstract Introduction: PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevent MDS leukemic transformation and progression. Alternative Polyadenylation (APA) regulation and 3' RNA cleavage are 2 known vital processes in cellular DNA damage response to maintain genomic stability. NUDT21 gene encodes protein that is one subunit of the cleavage factor Im complex required for 3' RNA cleavage and APA processing. Although PIGN gene has six potential polyadenylation sites, the exact role of NUDT21 product on PIGN expression regulation has not been illustrated before. Methods: At first, we analyzed NUDT21 gene expression profile in AML, MDS, and normal control specimens and to plot the relationship between NUDT21 gene expression status and patient survival length from 5 datasets. We also used correlation regression analysis method to predict the gene expression correlations between NUDT21 gene and PIGN gene. Then, we used Caspas9 technology and vector overexpression technology to knockout or overexpress PIGN in HEK293 cells; RNA interference to knockdown the NUDT21 gene in HEK293, HL60, K562, THP-1,SKM-1 and MUTZ-1 cells. MTT and CCK8 methods were used to evaluate cell proliferation. Meanwhile, cell cycle, apoptosis, Reactive Oxygen Species (ROS) and GPI-anchored proteins (CD55, CD59, and CD235) expression profiles were evaluated by flowcytometry; Immunofluorescence experiment, RT-PCR were used to evaluate the impact of NUDT21 gene knockdown on PIGN expression. in vivo mice siRNA interference tests were conducted to verify the impact of NUDT21 knockdown on PIGN gene expression. RNA-Seq & Dapars analyses were conducted on mRNAs from 3 cell lines with various PIGN gene expression status to observe the effects of NUDT21 knockdown on PIGN 3'UTR processing. Results: NUDT21 expression increases in AML and high-risk MDS. NUDT21 expression level is negatively correlated with overall survival. NUDT21 knockdown decreases cell proliferation but increases cell differentiation via prolonging G1 phase, decreasing S phase, and increasing apoptosis. NUDT21 knockdown increases cell membrane GPI-anchoring proteins (CD55, CD59, and CD235) expressions; increases ROS and Caspas3 expressions but decreases CyclinD1 expression. NUDT21 knockout can increase PIGN expression, but PIGN expression status has no impact on NUDT21 expression. RT-PCR and WB results showed that NUDT21 knockdown can increase TP53 expression but decrease p21 expression. in vivo mice siRNA interference tests confirmed that NUDT21 knockdown increases PIGN gene expression. The RNA-Seq/Dapars analyses results showed that 3'UTR of PIGN transcript was significantly shortened and PIGN expression increased after NUDT21 knockdown. Conclusion: NUDT21 modulates PIGN gene expression through APA mechanism. PIGN polyadenylation may regulate cell proliferation status via modulating cell cycle and apoptosis. Citation Format: Jeffrey J. Pu, Fanggang Ren, Na Zhang, Shuo Li, Lan Zhang, Eric Miller, Guirong Wang, Hongwei Wang. NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4741.

NUDT21 Modulates Pign through APA Mechanism to Affect the Progression of MDS and AML

Introduction: Previous studies indicated that PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevent MDS leukemic transformation and progression. Alternative Polyadenylation (APA) regulation and 3' RNA cleavage are 2 known vital processes in cellular DNA damage response to maintain genomic stability. NUDT21 gene encodes protein that is one subunit of the cleavage factor Im complex required for 3' RNA cleavage and APA processing. Although PIGN gene has six potential polyadenylation sites, the exact role of NUDT21 product on PIGN expression regulation has not been illustrated before. In this study, we assessed NUDT21 product regulation of PIGN expression via APA in MDS and AML patient samples, cell lines, and in mouse. Methods: At first, we used bioinformatics methods to analyze NUDT21 gene expression profile in AML, MDS, and normal control specimens and to plot the relationship between NUDT21 gene expression status and patient survival length from 5 datasets. We also used correlation regression analysis method to predict the gene expression correlations between NUDT21 gene and PIGN gene. Then, we used Caspas9 technology and vector overexpression technology to knockout or overexpress PIGN in HEK293 cells; RNA interference to knockdown the NUDT21 gene in HEK293, HL60, K562, THP-1,SKM-1 and MUTZ-1 cells. MTT and CCK8 methods were used to evaluate cell proliferation. Meanwhile, cell cycle, apoptosis, Reactive Oxygen Species (ROS) and GPI-anchored proteins (CD55, CD59, and CD235) expression profiles were evaluated by flowcytometry; Immunofluorescence experiment, RT-PCR were used to evaluate the impact of NUDT21 gene knockdown on PIGN expression. in vivo mice siRNA interference tests were conducted to verify the impact of NUDT21 knockdown on PIGN gene expression. RNA-Seq & Dapars analyses were conducted on mRNAs from THP-1,SKM-1 and MUTZ-1 cell lines with various PIGN gene expression status to observe the effects of NUDT21 knockdown on PIGN 3'UTR processing. Results:NUDT21 expression increases in mononuclear cells from AML and high-risk MDS. NUDT21 expression level is negatively correlated with the overall survival. NUDT21 knockdown decreases cell proliferation but increases cell differentiation via prolonging G1 phase, decreasing S phase, and increasing apoptosis. NUDT21 knockdown increases cell membrane GPI-anchoring proteins (CD55, CD59, and CD235) expressions; increases ROS and Caspas3 expressions but decreases CyclinD1 expression. NUDT21 knockout can increase the PIGN expression, but PIGN expression status has no impact on NUDT21 expression. RT-PCR and WB results showed that NUDT21 knockdown can increase TP53 expression but decrease p21 expression. in vivo mice siRNA interference tests confirmed that NUDT21 knockdown increases PIGN gene expression (Figure). The RNA-Seq/Dapars analyses results showed that 3'UTR of PIGN transcript was significantly shortened and PIGN expression increased after NUDT21 knockdown. Conclusion:NUDT21 modulates PIGN gene expression through APA mechanism. PIGN polyadenylation modulation may regulate cell cycle and cell proliferation through NUDT21-PIGN-P21- Cyclin D1 pathway and may regulate cell apoptosis through NUDT21-PIGN-TP53-ROS-caspas3 pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association.

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3′-UTR processing, we observed a high association of Star-PAP at the 3′-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3′-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Post-transcriptional regulation is involved in the cold-active methanol-based methanogenic pathway of a psychrophilic methanogen.

The methanol-derived methanogenetic pathway contributes to bulk methane production in cold regions, but the cold adaptation mechanisms are obscure. This work investigated the mechanisms using a psychrophilic methylotrophic methanogen Methanolobus psychrophilus R15. R15 possesses two mtaCB operon paralogues-encoding methanol:corrinoid methyltransferase that is key to methanol-based methanogenesis. Molecular combined methanogenic assays determined that MtaC1 is important in methanogenesis at the optimal temperature of 18°C, but MtaC2 can be a cold-adaptive paralogue by highly upregulated at 8°C. The 5'P-seq and 5'RACE all assayed that processing occurred at the 5' untranslated region (5'-UTR) of mtaC2; reporter genes detected higher protein expression, and RNA half-life experiments assayed prolonged lifespan of the processed transcript. Therefore, mtaC2 5'-UTR processing to move the bulged structure elevated both the translation efficiency and transcript stability. 5'P-seq, quantitative RT-PCR and northern blot all identified enhanced mtaC2 5'-UTR processing at 8°C, which could contribute to the upregulation of mtaC2 at cold. The R15 cell extract contains an endoribonuclease cleaving an identified 10nt-processing motif and the native mtaC2 5'-UTR particularly folded at 8°C. Therefore, this study revealed a 5'-UTR processing mediated post-transcriptional regulation mechanism controlling the cold-adaptive methanol-supported methanogenetic pathway, which may be used by other methylotrophic methanogens.

A Splicing-Independent Function of RBM10 Controls Specific 3′ UTR Processing to Regulate Cardiac Hypertrophy

RNA binding motif protein 10 (RBM10) is a regulator ofalternative splicing in apoptosis and inflammation. We discovered a splicing-independent function of RBM10 critical for the regulation of heart failure (HF). RBM10 is enriched in the heart and associates with Star-PAP (TUT1) to control the expression and 3' end processing of cardiac mRNAs. The RBM10 RRM2 domain binds the Star-PAP catalytic domain, which directs Star-PAP activity toward polyadenylation. RBM10 binds the pre-mRNA UTR, assembles the Star-PAP complex, and guides this complex specifically to mRNAs encoding anti-hypertrophy regulators.Accordingly, we tested cellular hypertrophy in rat cardiomyoblasts and cardiac hypertrophy (CH) and the subsequent progression to HF in Wistar rat hearts. We demonstrated downregulation of RBM10 during CH and HF. Ectopic re-expression of RBM10 rescued cardiomyocyte hypertrophy. RBM10 depletion evoked a hypertrophic response in H9c2 cells. Our results establish an anti-hypertrophy mechanism mediated by RBM10 in the heart that is directly linked to HF.

Alpha-synuclein mRNA isoform formation and translation affected by polymorphism in the human SNCA 3'UTR.

BackgroundMultiple variants in SNCA, encoding alpha‐synuclein, a main component of Lewy bodies, are implicated in Parkinson's disease.MethodsWe searched for cis‐acting SNCA variants using allelic mRNA ratios in human brain tissues. In a SNCA 3′UTR (2,520 bp) luciferase reporter gene assay, translation in SH‐SY5Y cells in the presence of the rs17016074 G/A alleles was measured. To assess clinical impact, we queried neurocognitive genome‐wide association studies.ResultsAllelic ratios deviated up to twofold, measured at a marker SNP in the middle of a long 3′ untranslated region (3′UTR), but not at a marker at its start, suggesting regulation of 3′UTR processing. 3′UTR SNP rs17016074 G/A, minor allele frequency (MAF) <1% in Caucasians, 13% in Africans, strongly associates with large allelic mRNA expression imbalance (AEI), resulting in reduced expression of long 3′UTR isoforms. A second 3′UTR SNP (rs356165) associates with moderate AEI and enhances SNCA mRNA expression. The rs17016074 A allele reduces overall 3′UTR expression in luciferase reporter gene assays but supports more efficient translation, resolving previous contradictory results. We failed to detect significant genome‐wide associations for rs17016074, possibly a result of low MAF in Caucasians or its absence from most genotyping panels. In the “Genome Wide Association Study of Yoruba in Nigeria,” rs356165 was associated with reduced memory performance.ConclusionsHere, we identify two cis‐acting regulatory variants affecting SNCA mRNA expression, measured by allelic ratios in the 3′UTR. The rs17016074 minor A allele is associated with higher expression of luciferase protein activity. Resolving the genetic influence of SNCA polymorphisms requires study of the interactions between multiple regulatory variants with distinct frequencies among populations.

The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis.

The epitranscriptomic mark N6-methyladenosine (m6A) can be written, read, and erased via the action of a complex network of proteins. m6A binding proteins read m6A marks and transduce their downstream regulatory effects by altering RNA metabolic processes. The characterization of m6A readers is an essential prerequisite for understanding the roles of m6A in plants, but the identities of m6A readers have been unclear. Here, we characterized the YTH-domain family protein ECT2 as an Arabidopsis thaliana m6A reader whose m6A binding function is required for normal trichome morphology. We developed the formaldehyde cross-linking and immunoprecipitation method to identify ECT2-RNA interaction sites at the transcriptome-wide level. This analysis demonstrated that ECT2 binding sites are strongly enriched in the 3' untranslated regions (3' UTRs) of target genes and led to the identification of a plant-specific m6A motif. Sequencing analysis suggested that ECT2 plays dual roles in regulating 3' UTR processing in the nucleus and facilitating mRNA stability in the cytoplasm. Disruption of ECT2 accelerated the degradation of three ECT2 binding transcripts related to trichome morphogenesis, thereby affecting trichome branching. The results shed light on the underlying mechanisms of the roles of m6A in RNA metabolism, as well as plant development and physiology.

Discovery of physiological and cancer-related regulators of 3\u2032 UTR processing with KAPAC

3′ Untranslated regions (3' UTRs) length is regulated in relation to cellular state. To uncover key regulators of poly(A) site use in specific conditions, we have developed PAQR, a method for quantifying poly(A) site use from RNA sequencing data and KAPAC, an approach that infers activities of oligomeric sequence motifs on poly(A) site choice. Application of PAQR and KAPAC to RNA sequencing data from normal and tumor tissue samples uncovers motifs that can explain changes in cleavage and polyadenylation in specific cancers. In particular, our analysis points to polypyrimidine tract binding protein 1 as a regulator of poly(A) site choice in glioblastoma.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.

The C421A (Q141K) polymorphism enhances the 3′-untranslated region (3′-UTR)-dependent regulation of ATP-binding cassette transporter ABCG2

The impact of the gout-causing C421A (Q141K) single nucleotide polymorphism (SNP) on ABC transporter ABCG2 expression and function has been extensively characterized. However, the influence of the C421A SNP on 3′-UTR-dependent ABCG2 regulation has not been analysed so far. To elucidate this matter, we generated vectors for expression of either the ABCG2 coding sequence (ORF) or the ABCG2 ORF fused to its 3′-UTR, inserted the C421A mutation via site-directed mutagenesis and expressed wild-type and C421A-mutated ABCG2 transcripts in HEK293-Tet-On cells. As shown previously, the C421A SNP significantly reduced ABCG2 protein levels in ABCG2 ORF-transfected HEK293-Tet-On cells. Interestingly, the presence of the 3′-UTR in the ABCG2 transcript dramatically reduced ABCG2 protein content in cells transfected with the C421A variant but not significantly in those transfected with ABCG2 wild-type sequence, whereas ABCG2 mRNA levels were similar. siRNA-mediated DICER1 knockdown to reduce cellular microRNA biogenesis and selective mutation of putative microRNA binding sites within the ABCG2 3′-UTR partially antagonized C421A-associated reduction of ABCG2 protein content but did not significantly affect wild-type ABCG2 protein levels. In addition, antagomir-mediated inhibition of two microRNAs (hsa-miR-519c and hsa-miR-328) again partially reversed C421A-associated ABCG2 translational repression, thereby indicating that the C421A SNP may facilitate microRNA-dependent repression of ABCG2 protein translation. We conclude from our results that the C421A SNP may lead to reduced ABCG2 protein levels not only by affecting cellular protein stability but also via enhanced microRNA-dependent ABCG2 repression. Moreover, tissue-specific variation in ABCG2 3′-UTR processing may profoundly affect ABCG2 expression levels in individuals carrying the C421A mutation.

CPEB1 coordinates alternative 3'-UTR formation with translational regulation.

More than half of mammalian genes generate multiple messenger RNA isoforms that differ in their 3' untranslated regions (3' UTRs) and therefore in regulatory sequences, often associated with cell proliferation and cancer; however, the mechanisms coordinating alternative 3'-UTR processing for specific mRNA populations remain poorly defined. Here we report that the cytoplasmic polyadenylation element binding protein 1 (CPEB1), an RNA-binding protein that regulates mRNA translation, also controls alternative 3'-UTR processing. CPEB1 shuttles to the nucleus, where it co-localizes with splicing factors and mediates shortening of hundreds of mRNA 3' UTRs, thereby modulating their translation efficiency in the cytoplasm. CPEB1-mediated 3'-UTR shortening correlates with cell proliferation and tumorigenesis. CPEB1 binding to pre-mRNAs not only directs the use of alternative polyadenylation sites, but also changes alternative splicing by preventing U2AF65 recruitment. Our results reveal a novel function of CPEB1 in mediating alternative 3'-UTR processing, which is coordinated with regulation of mRNA translation, through its dual nuclear and cytoplasmic functions.

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability, processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3'-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3'-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3'-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3'-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3'-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3'-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3'-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3'-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3'-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.

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.

A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR

The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3′-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

A new in vitro translation system for non‐radioactive assay from tobacco chloroplasts: effect of pre‐mRNA processing on translation in vitro

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3′ untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3′ untranslated region (3′UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3′UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3′UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca2+-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3′-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (Bmax) 0.25 time for petD 3′UTR, and three times for psbA 3′UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that Bmax, not Kd, might be a more important factor in the P-24RNP-3′UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.

Effects of splitting alternative KlCYC1 3′-UTR regions on processing: Metabolic consequences and biotechnological applications

To analyze the functionality of alternative 3′-UTR processing in the yeast Kluyveromyces lactis, recombinant forms of the KlCYC1 gene containing the proximal (1–713) or the distal (699–1194) 3′-UTR region (positions related to the TAA stop codon) were obtained. The cells expressing the gene with proximal 3′-UTR showed the same growth phenotype as the wild type. When the gene expressed only the distal region, a single transcript was generated and its expression was increased in late-growth phases. Cells expressing the alternative distal 3′-UTR region showed differences in their levels of cytochrome c biomass and ethanol production with respect to the wild type. The split 3′-UTR regions were also functional as separate processing units in Saccharomyces cerevisiae. The importance of our results in recombinant gene expression applications will be discussed.

The KlCYC1 gene, a downstream region for two differentially regulated transcripts.

KlCYC1 encodes for cytochrome c in the yeast Kluyveromyces lactis and is transcribed in two mRNAs with different 3'-processing points. This is an uncommon transcription mechanism in yeast mRNAs. The 3' sequence encompassing the whole region that is needed to produce both mRNAs is analysed. We have determined identical processing points in K.lactis and in Saccharomyces cerevisiae cells transformed with KlCYC1; positions 698 and 1092 (with respect to the TAA) are the major polyadenylation points. This shows that the cis-elements present in the KlCYC1 3'-untranslated region (3'-UTR) direct a processing mechanism that has been conserved in yeast. In K. lactis there is a high predominance of the shorter transcript (1.14 kb) only at the initial logarithmic growth phase. Interestingly, this growth phase-dependent regulation of 3'-UTR processing is lost when the gene is expressed in S. cerevisiae.