Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals.

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.

A Foundational Study for Normal F8-Containing Mouse Models for the miRNA Regulation of Hemophilia A: Identification and Analysis of Mouse miRNAs that Downregulate the Murine F8 Gene.

Hemophilia A (HA) is associated with defects in the F8 gene, encoding coagulation factor VIII (FVIII). Our previous studies show that F8-targeting micro RNAs (miRNAs), a group of small RNAs involved in gene regulation, can downregulate F8 expression causing HA in individuals with normal F8-genotypes and increased HA severity in patients with mutations in F8. Understanding the mechanistic underpinnings of human genetic diseases caused or modulated by miRNAs require a small animal model, such as a mouse model. Here, we report a foundational study to develop such a model system. We identified the mouse 3'untranslated region (3'UTR) on murine F8-mRNA (muF8-mRNA) that can bind to murine miRNAs. We then selected three miRNAs for evaluation: miR-208a, miR-351 and miR-125a. We first demonstrate that these three miRNAs directly target the 3'UTR of muF8-mRNA and reduce the expression of a reporter gene (luciferase) mRNA fused to the muF8-3' UTR in mammalian cells. Furthermore, in mouse cells that endogenously express the F8 gene and produce FVIII protein, the ectopic expression of these miRNAs downregulated F8-mRNA and FVIII protein. These results provide proof-of-concept and reagents as a foundation for using a normal F8-containing mouse as a model for the miRNA regulation of normal F8 in causing or aggravating the genetic disease HA.

MicroRNA-223 Regulates Septin-2 and Septin-6 in Stored Platelets.

BACKGROUND: Septins have been identified to play important roles in platelets, but their regulation in platelets is unknown. Human platelet being an enucleated and terminally differentiated cell, mRNA downregulation by miRs is one of the posttranscriptional mechanisms operative in platelets. OBJECTIVE: Since platelets are known to have miR-223 in abundance, the objective of this study is to test whether a) platelet septins have miR-223 interacting target sites in their mRNA 3'UTRs, b) septin mRNAs and miR-223 form complexes with Argonaute 2 (AGO2) protein in platelets, which is the catalytic component of an RNA Induced Silencing Complex (RISC), c) a reporter gene with septin mRNA 3' untranslated region (UTR) is subjected to downregulation by miR-223 and d) anti-miR-223 can suppress miR-223 activity and enhance septin-2 expression in platelets. METHOD: Bioinformatics tools were used to screen mRNA 3'UTRs of septin-2 and septin-6 for miR- 223 target sites. Subsequently, platelet extracts were immunoprecipitated by AGO2 antibodies to identify that the two septin mRNAs and miR-223 were in complex with AGO2. A luciferase reporter chimeric- gene expression system was utilized to monitor miR-223 mediated downregulation luciferase gene containing the 3'UTR of either septin-2 or septin-6. Further, anti-miR-223 was utilized in platelets to directly demonstrate the role of miR-223 on the expression of septin-2. RESULTS: Our results demonstrate that in stored platelets a) septine-2 and septin-6 mRNAs have miR- 223 target sites, b) septin-2 and septin-6 are in complex with Ago-2, c) in luciferase reporter gene system, the interaction of miR-223 with 3' UTRs of septin-2 and septin-6 leads to downregulation of luciferase expression and d) anti-miR-223 downregulated miR-223 activity and thereby the expression of septin-2 is upregulated. CONCLUSION: The results demonstrate that like in nucleated cells, enucleated platelets also have miRbased mechanisms for the regulation of their septins.

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.

An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations.

UNLABELLED: Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3' end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes. IMPORTANCE: Ribosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin and that this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3' end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough.

Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3' Untranslated Region.

UNLABELLED: Turnip crinkle virus (TCV) contains a structured 3' region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3' untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3'UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCE: Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3' UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3' UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.

Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA.

The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5' sequences from a second TCV satRNA (satD) and 3' sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5' end of satC.

Position of the kissing-loop interaction associated with PTE-type 3'CITEs can affect enhancement of cap-independent translation.

The Panicum mosaic virus-like translation enhancer (PTE) functions as a cap-independent translation enhancer (3'CITE) in members of several Tombusviridae genera including 7/19 carmoviruses. For nearly all PTE, a kissing-loop connects the element with a hairpin found in several conserved locations in the genomic RNA (5' terminal hairpin or ~100 nt from the 5' end) and small subgenomic RNA (~63 nt from the 5' end). Moving the interaction closer to the 5' end in reporter mRNAs using Saguaro cactus virus (SCV) sequences had either a minimal or substantial negative effect on translation. Movement of the kissing loop from position 104 to the SCV 5' terminal hairpin also reduced translation by 4-fold. These results suggest that relocating the PTE kissing loop closer to the 5' end reduces PTE efficiency, in contrast to results for the Barley yellow dwarf BTE and Tomato bushy stunt virus Y-shaped 3'CITEs, suggesting that different 3'CITEs have different bridging requirements.

A comparative study of the Arabidopsis thaliana guard-cell transcriptome and its modulation by sucrose.

Microarray analysis was performed on RNA isolated from guard cells that were manually dissected from leaves of Arabidopsis. By pooling our data with those of two earlier studies on Arabidopsis guard cell protoplasts, we provide a robust view of the guard-cell transcriptome, which is rich in transcripts for transcription factors, signaling proteins, transporters, and carbohydrate-modifying enzymes. To test the hypothesis that photosynthesis-derived sugar signals guard cells to adjust stomatal opening, we determined the profile of genes expressed in guard cells from leaves that had been treated with sucrose. The results revealed that expression of 440 genes changed in guard cells in response to sucrose. Consistent with this hypothesis, these genes encoded cellular functions for photosynthesis and transport of sugars, water, amino acids, and ions. Plants of T-DNA insertion lines for 50 genes highly responsive to sucrose were examined for defects in guard cell function. Twelve genes not previously known to function in guard cells were shown to be important in leaf conductance, water-use efficiency, and/or stomate development. Of these, three are of particular interest, having shown effects in nearly every test of stomatal function without a change in stomatal density: TPS5 (At4g17770), a TRAF domain-containing protein (At1g65370), and a WD repeat-containing protein (At1g15440).

Long-distance kissing loop interactions between a 3' proximal Y-shaped structure and apical loops of 5' hairpins enhance translation of Saguaro cactus virus.

Circularization of cellular mRNAs is a key event prior to translation initiation. We report that efficient translation of Saguaro cactus virus (SCV) requires a 3' translational enhancer (PTE) located partially in coding sequences. Unlike a similar PTE reported in the 3' UTR of Pea enation mosaic virus that does not engage in an RNA:RNA interaction (Wang Z. et al., J. Biol. Chem. 284, 14189-14202, 2009), the SCV PTE participates in long distance RNA:RNA interactions with hairpins located in the p26 ORF and in the 5' UTR of one subgenomic RNA. At least two additional RNA:RNA interactions are also present, one of which involves the p26 initiation codon. Similar PTE can be found in six additional carmoviruses that can putatively form long-distance interactions with 5' hairpins located in comparable positions.