Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantitate the synthesis of bovine coronavirus (BCoV) defective interfering (DI) RNA (−) strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3′-terminal 55 nucleotides (nts) which function in the synthesis of (−)- or (+)-strand BCoV DI RNA. The major findings are as follows: (i) nts from -5 to -39 within the 3′-terminal 55 nts are the cis-acting elements responsible for (−)-strand BCoV DI RNA synthesis, (ii) nts from −3 to −34 within the 3′-terminal 55 nts are cis-acting elements required for (+)-strand BCoV DI RNA synthesis, and (iii) the nucleotide species at the 3′-most position (−1) is important, but not critical, for both (−)- and (+)-strand BCoV DI RNA synthesis. These results demonstrate that the 3′-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (−)- and (+)-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (−)-strand RNA synthesis in coronaviruses.The synthesis of the negative-strand [(−)-strand] complement of the ∼30 kilobase, positive-strand [(+)-strand] coronaviral genome is a necessary early step for genome replication. The identification of  After entry into a cell, the positive-strand [(+)-strand] RNA virus genome first serves as a template for synthesis of viral proteins required for subsequent virus replication. The synthesis of the negative-strand [(−)-strand] complement from the (+)-strand genome is the first step of genome replication and is assumed to be initiated from the 3′ end of the (+)-strand RNA genome. The 3′ end of the synthesized (−)-strand RNA genome is then employed as the site for the initiation of the (+)-strand RNA genome. Therefore, the RNA elements on the 3′ end of (+)- and (−)-strand RNA genome may play critical roles in targeting the viral replication complex to initiate the synthesis of their counterparts.Coronavirinae, the family Coronaviridae and the order Nidovirales, belongs to the genus betacoronaviruses, which includes the mouse hepatitis coronavirus (MHV) cis-acting RNA elements in the coronavirus genome required for replication [interpreted as (+)-strand RNA synthesis] have been identified in the past two decades with a full-length infectious cDNA or coronavirus defective interfering (DI) RNA, a surrogate for the coronavirus genome cis-acting RNA elements in the 3′ UTR of coronavirus, it has been demonstrated that the minimal requirement for replication maps 436 nucleotides (nts) from the 3′ end of MHV DI RNA according to deletion analyses cis-acting RNA elements that are essential for coronavirus replication, including a 61-nt bulged stem-loop in MHV Bovine coronavirus (BCoV), a (+)-strand RNA virus in the subfamily cis-acting RNA elements required for (−)-strand RNA synthesis in coronaviruses have been hampered by the limit amount of (−)-strand RNA present in the virus-infected cells and the lack of sufficient methods to detect (−)-strand coronaviral RNA in low numbers. For example, there are only an estimated ∼5 molecules of BCoV DI RNA (−) strand per cell in BCoV DI RNA-transfected BCoV-infected human rectal tumor (HRT)-18 cells at 24 hour postinfection (hpi) of virus passage 1 (VP1) cis-acting elements for the synthesis of BCoV (−)-strand DI RNA.In contrast, studies on the cis-acting elements within the 3′-terminal 55 nts that are required for the synthesis of (+)- or (−)-strand BCoV DI RNA by simultaneous comparison of their efficiency of RNA synthesis. Our findings suggest that the 3′-terminal 55 nts harbor cis-acting RNA elements required for both (+)- and (−)-strand RNA synthesis.In this study we used head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantitate BCoV (−)-strand DI RNA synthesis. Using this technique along with Northern blot assay, we specifically defined the cis-acting elements in the 3′ UTR of BCoV and MHV-A59 have been characterized cis-acting elements critical for coronavirus replication.Before the outbreak of severe acute respiratory syndrome (SARS), betacoronaviruses were considered to include one lineage. After the discovery of SARS-CoV, other novel subgroups of the betacoronaviruses have been proposed and the original group 2 coronaviruses, which included BCoV and MHV, became designated as 2a coronaviruses cis-acting elements required for coronavirus replication cis-acting RNA elements that are essential for the (−)-strand DI RNA synthesis in coronaviruses, however, has been restricted by the low numbers of (−)-strand DI RNA produced from the DI RNA-transfected helper virus-infected cells in vitro using plasmid DNA as a template The coronavirus DI RNA has been employed as a surrogate for coronavirus genome to study the cis-acting RNA elements required for (–)- and (+)-strand DI RNA synthesis within this 55-nt region, however, remain to be determined. To define the specific cis-acting RNA elements within the 3′-terminal 55 nts which function in the synthesis of BCoV DI RNA (–) or (+) strands, a series of DI RNA constructs with deletions either from the 3′ or 5′ end of the 55-nt region were generated by mutagenesis (We have demonstrated in the present study that the 3′-most 55 nts are required for the synthesis of (–)-strand BCoV DI RNA using the head-to-tail RNA ligation and qRT-PCR . The mapagenesis  and testagenesis . The totagenesis  althoughagenesis  from theagenesis  and (+)-cis-acting RNA motif required for (–)- and (+)-strand RNA synthesis within this 55-nt region, DI RNA constructs with various deletions from the 5′ terminus of this region were created and tested - or (+)-strand synthesis may reside in the region between the nts –2 and –40. To further determine the requirement of cis-acting elements for the (–)- and (+)-strand BCoV DI RNA synthesis in this region, deletion mutants Δ5–14, Δ15–24, Δ25–34, and Δ35–39 were constructed (cis-acting elements responsible for the (−)-strand BCoV DI RNA synthesis and that the nts from −3 to −34 within the 3′-terminal 55 nts are the cis-acting elements required for the (+)-strand BCoV DI RNA synthesis.To further dissect the specific d tested . The effd tested  althoughd tested , lane 10d tested  but stild tested , lane 12d tested  and no -strand RNA synthesis in coronaviruses. Since the last nucleotide of the 3′ UTR in all coronavirus genomes sequenced to date is cytosine, it was speculated that this conserved nucleotide species may play an important role in coronavirus replication. To test this hypothesis, the cytosine residing at the last nucleotide of the 3′ UTR in BCoV DI RNA was substituted with adenine, uracil and guanine to make the constructs BM-3′A, BM-3′U, and BM-3′G, respectively . As showcis-acting elements required for coronavirus replication have been identified in the past two decades cis-acting elements specific for (−)-strand RNA synthesis in coronavirus, however, has been hampered due to insufficiencies in the techniques used to detect and quantitate RNA (−) strand species. In this study, we employed the method of head-to-tail ligation and qRT-PCR to examine the cis-acting elements required for (−)-strand BCoV DI RNA synthesis cis-acting RNA elements required for (−)-strand DI RNA synthesis and to gain insight into the mechanism of coronavirus replication.Many cis-acting elements in coronaviruses emphasize their roles in replication [interpreted as (+)-strand synthesis]; however, it is still unclear that the identified cis-acting elements function in (−)- or (+)-strand synthesis. We in this study, therefore, simultaneously analyzed the effects of cis-acting elements on the synthesis of both (−)- and (+)-strand BCoV DI RNA during infection in order to specifically define the role of a cis-acting element. According to the results concerning the (−)- and (+)- strand DI RNA synthesis from mutants Δ5, Δ15, Δ30 and Δ55, we conclude that the deleted sequences may function at least in the (−)-strand DI RNA synthesis because the deleted sequences significantly blocked the (−)-strand DI RNA synthesis from their (+)-strand DI RNA templates (cis-acting elements in the synthesis of (+)-strand rather than (−)-strand DI RNA. In comparison with wt BM25A, the efficiency of both (−)- and (+)-strand DI RNA synthesis from the mutants Δ55–45 and Δ55–40 was not significantly inhibited, suggesting nts from −55 to −40 are not absolutely required for both (−)- and (+)-strand DI RNA synthesis. Interestingly, the efficiency of (−)-strand RNA synthesis from deletion mutant Δ55-45 was even higher than that from wt BM25A as shown in cis-acting element [on (−)-strand DI RNA] required for (+)-strand DI RNA synthesis is intact. These results are also in agreement with the previously proposed model which indicated that the (−)-strand viral RNA is repeatedly used as a template for (+)-strand viral RNA synthesis cis-acting elements that function in (−)- or (+)-strand DI RNA synthesis.Replication of (+)-strand RNA viruses can be generally divided into two steps. First, the viral polymerase employs a (+)-strand genome as a template to generate the (−)-strand complement. Second, the synthesized (−)-strand genome then serves as a template for the synthesis of the (+)-strand counterpart. The majority of previous studies on emplates . Regardiemplates . HoweverBecause the last nucleotide of the 3′ UTR in all coronavirus genomes sequenced to date is cytosine and coronavirus nonstructural protein 8 has been proposed to preferentially initiate the synthesis of primer at the cytosine within the internal 5′-(G/U)CC-3′ trinucleotides cis-acting elements identified in this study and others are involved in the constitution of replication complex in the (−)-strand RNA genome, which is required for the subsequent (+)-strand RNA synthesis.The initiation of (−)-strand RNA synthesis in (+)-strand RNA viruses occurs at the 3′ terminal sequences of (+)-strand RNA, and consequently alterations of the 3′-terminal sequences of (+)-strand RNA may be detrimental to (−)-strand RNA synthesis as evidenced by this study and others cis-acting elements in the (−)- or (+)-strand DI RNA synthesis. In coronaviruses, besides the cis-acting elements identified here, many cis-acting elements located in the 5′ UTR, 3′ UTR and coding region have been demonstrated to be required for replication; however, their specific function in (−)- or (+)-strand RNA synthesis remains to be determined. Using the method may contribute to elucidate the specific roles of these cis-acting elements.In this study we performed the head-to-tail ligation of RNA and qRT-PCR to detect and quantitate coronavirus (−)-strand DI RNA synthesis. The method, along with Northern blot assay, allows us to simultaneously detect both (−)- and (+)-strand RNA synthesis and specifically define the functions of 7 PFU/ml was used as a helper virus in human rectal tumor (HRT)-18 cell line as described previously A DI RNA-free stock of the Mebus strain of BCoV (GenBank accession no. U00735) at 3×10SpeI and MluI. The digested fragment was cloned into SpeI- and MluI-linearized pDI RNA-1 to create pBM25A containing MHV-A59 3′ UTR and 25-nt poly(A) tail.To generate the pBM25A construct, PCR was performed with the oligonucleotides TGEV 7(−) and BM25A(+) and with pDI RNA-1 in which the 288-nt 3′ UTR of BCoV-Mebus in pDrep1 was replaced with the 301-nt 3′ UTR and 43-nt poly(A) tail of MHV-A59 (GenBank accession no. NC_001846) as a template SpeI and MluI was cloned into SpeI- and MluI-linearized pBM25A to make the mutant pΔ55. Mutants of pΔ1, pΔ2, pΔ3, pΔ4, pΔ5, pΔ15, pΔ30, pΔ55–45, pΔ55–40, pΔ55–35, pΔ55–30, pΔ30–15, pΔ5–14, pΔ15–24, pΔ25–34, pΔ35–39, pBM-3′A, pBM-3′G, and pBM-3′U were similarly constructed, except for the corresponding oligonucleotides used in the first and second reactions as described in To construct the mutant pΔ55, an overlap PCR mutagenesis procedure was performed as previously described in vitro, the MluI-linearized plasmid DNA construct was transcribed with the RiboMAX Large Scale RNA Production System-T7 (Promega) according to the manufacturer's instructions and was chromatographed through a Biospin 6 column (Bio-Rad) before use. To analyze the synthesis of the (–)-strand DI RNA (5 cells/dish) were infected with BCoV at a multiplicity of infection of 5 PFU per cell. After 2 hours of infection, 3 µg of BM25A or mutant transcript quantitated by RNA denaturing gel was transfected into BCoV-infected HRT-18 cells. The total cellular RNA was extracted with TRIzol (Invitrogen) at 8 hpt, and 10 µg of extracted RNA in 25 µl of water, 3 µl of 10X buffer and 10 U of (in 1 µl) tobacco acid pyrophosphatase (TAP) (Epicentre) were used to remove the 5′-triphosphate groups of the (–)-strand DI RNA, generating monophosphate DI RNA (–) strand. Following the treatment of TAP, phenol-chloroform-extracted RNA in 25 µl of water was heat-denatured at 95°C for 5 min and then quickly cooled. Then, 3 µl of 10X ligase buffer and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were added, and the mix was incubated for 16 h at 16°C. Phenol-chloroform-extracted ligated RNA was quantitated, and 1 µg of ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–), which binds nts 89–112 from the poly(U) tail in the (–) strand of the MHV-A59 3′ UTR, to synthesize cDNA with SuperScript III reverse transcriptase (Invitrogen). Of this, 5 µl was used in a 50-µl PCR with AccuPrime Tag DNA polymerase (Invitrogen) and oligonucleotide MHV3′UTR6(–), which binds nts 64-85 from the poly(U) tail in the (–) strand of the MHV-A59 3′ UTR, and BCV23-40(+), which binds nts 23–40 in the (+)-strand leader sequence of BCoV. The mixture was heated to 94°C for 2 min and then subjected to 50 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. The ∼150- bp PCR product was sequenced after cloning into TOPO XL vector (Invitrogen).To synthesize transcripts d DI RNA , the HRT8 to 10 copies of each plasmid). In addition, 18S rRNA with primers 18S rRNA(–) and 18S rRNA(+), DI RNA with primers MHV3UTR2(–) and BCV29-54(+) and N sgmRNA with primers leader20(–) and BCVN(+) were applied as internal controls for normalization of (–)-strand DI RNA synthesis. The reactions were conducted with an initial pre-incubation at 95°C for 5 min, followed by 35 amplification cycles as follows: 95°C for 15 s and 60°C for 60 s.For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen). Real-time PCR amplification using the primers MHV3′UTR6(–) and BCV23-40(+) was performed using TagMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer recommendations with a LightCycler 480 instrument (Roche Applied Science). To quantitate the synthesis of (–)-strand DI RNA, dilutions of plasmids containing the same sequence as the detected RT-PCR product of (–) –strand DI RNA were always run in parallel with quantitated cDNA for use as standard curves  (for DI RNA), BCVN(+) (for N sgmRNA) or 18SrRNA(+) (for 18S rRNA), which was tailed with digoxigenin (DIG)-ddUTP using a DIG Oligonucleotide 3′-End Labeling kit . The RNA detected was visualized according to the manufacturer's recommended procedure.To identify the sequence of the (–)-and (+)-strand DI RNA at 8 hpt and at 48 hpi of VP1, 10 µg of total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre) and ligated with T4 RNA ligase I (New England Biolabs). One microgram of ligated RNA was used for an RT reaction with oligonucleotide MHV 3′ UTR3(–) [for (–)-strand DI RNA] or BCV107(+) [for (+)-strand DI RNA] to synthesize cDNA with SuperScript III reverse transcriptase (Invitrogen), after which the oligonucleotides MHV 3′ UTR2(–) and BCV29-54(+) were used for PCR with AccuPrime Tag DNA polymerase (Invitrogen). The resultant PCR product was directly sequenced.Figure S1Schematic diagram of the procedure to detect the synthesis of both (−)- and (+)-strand DI RNA. The HRT-18 cells were infected with helper virus BCoV and then transfected with DI RNA. The virus contained within transfected cells was referred to as virus passage 0 (VP0). The total intracellular RNA was collected at 8 hpt to detect the synthesis of (−)-strand DI RNA with qRT-PCR. The supernatant collected at 48 hpt from the BCoV-infected DI RNA-transfected cells contained virus passage 1 (VP1) and was used to infect fresh HRT-18 cells. The total intracellular RNA collected at 48 hpi of VP1 was used to detect the synthesis of (+)-strand DI RNA with Northern blot assay. The 30-nt TGEV reporter is the binding site for the probe TGEV8(+) used in Northern blot assay.(TIF)Click here for additional data file.Figure S2The synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 24 hpi of VP1 and was analyzed by Northern blot assay with N sgmRNA and 18S rRNA used as internal controls. The sequence of the BCoV DI RNA at 24 hpi of VP1 was determined by direct sequencing of RT-PCR products. wt: wild type, mut: mutant, mx: mixed.(TIF)Click here for additional data file.Table S1Oligonucleotides used for this study.(TIF)Click here for additional data file.


The PA protein of Influenza A virus (IAV) encoded by segment 3 acts as a specialized RNA endonuclease in the transcription of the viral genome. The same genomic segment encodes for a second shorter protein, termed PA-X, with the first 191 N-terminal aminoacids (aa) identical to PA, but with a completely different C-ter domain of 61 aa, due to a ribosomal frameshifting. In addition, it has been shown that several IAV isolates encode for a naturally truncated PA-X variant, PAXΔC20, missing the last 20 aa. The biochemical properties of PA-X and PAXΔC20 have been poorly investigated so far. Here, we have carried out an enzymatic characterization of PA-X and its naturally deleted form, in comparison with PA from the human IAV strain A/WSN/33 (H1N1). Our results showed, to the best of our knowledge for the first time, that PA-X possesses an endonucleolytic activity. Both PA and PA-X preferentially cut single stranded RNA regions, but with some differences. In addition, we showed that PAXΔC20 has severely reduced nuclease activity. These results point to a previously undetected role of the last C-ter 20 aa for the catalytic activity of PA-X and support distinct roles for these proteins in the viral life cycle. Among tand Ni2+ ,10.Recently, through the analysis of 1300 sequences derived from different isolates, a new highly conserved open reading frame has been discovered, within the same genomic segment coding for the PA subunit, called ORF-X . The ORFSince the binding sites of PA for the PB1 subunit are not present in the C-ter region of PA-X, it has been hypothesized that the latter may be an accessory protein acting independently from the RNA polymerase. Cellular studies suggested its involvement in the degradation of host mRNAs, contributing in this way to the modulation of the viral pathogenesis ,12. To dPA-X is conserved among influenza viruses : it is pIn this study, by using recombinantly expressed PA, PA-X and PAXΔC20 proteins from the A/WSN/33 (H1N1) human influenza virus, we show, to the best of our knowledge for the first time, that PA-X possesses a robust endonucleolytic activity. Both PA and PA-X preferentially target ssRNA regions, but with some differences, depending on the structure of the RNA to be cleaved. In addition, we characterize for the first time the PAXΔC20 natural variant, showing that it possesses reduced endonucleolytic activity, when compared to PA-X full length. Collectively, our results indicate an important functional role of the last C-ter 20 aa of PA-X and support the idea of distinct roles of PA, PA-X and PAXΔC20 in the viral life cycle.32P] used for RNA labelling was purchased from Hartmann Analytic GmbH . The inhibitor of PA endonuclease, 2,4-dioxo-4-phenylbutanoic acid (DPBA), was purchased from MolPort . All other reagents were purchased from VWR International  or Sigma-Aldrich . ATP (500 μCurie) at final concentration of 25 μM. The 3′ and 5′ ends of the 38mer oligonucleotide corresponds to the vRNA ends with a short linker (All RNA oligonucleotides were purchased by Biomers with a 5′ end fluorescein (FAM) labelling, with exception of the 56mer that bears a 3′ end FAM modification, and of r(A)t linker .The different RNA oligonucleotides were hybridized to generate the various substrates, as summarized in Table 2, MnCl2, CoCl2, CaCl2 (as indicated). Endonuclease reactions were incubated at 37°C for 40 min (unless otherwise indicated in the figure legends) and reactions were stopped by adding 98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol FF, 0.025% bromophenol blue. Samples were heated at 95°C for 5 min and loaded on 7 M 12% polyacrylamide gel, run at 40 W in TBE 1X. The bands corresponding to degradation products were visualized by laser scanning densitometry on a Typhoon Trio  phosphorimager and quantified using the program ImageQuant. Due to the intrinsic sensitivity of RNA to spontaneous hydrolysis and RNase action, some degradation of the RNA substrates in the control reactions was always present, even in the presence of RNase inhibitors. During quantification, we have subtracted this degradation as background, from the bands of interest.Endonuclease assays were performed using the RNA substrates reported in Scheme Km and Vmax values, time course experiments were performed in duplicate under the assay conditions described above, in the presence of increasing RNA substrate concentrations. Four different substrate concentrations were used for each set of experiments. Data were subjected to linear interpolation and the slope of each curve was taken as the apparent reaction rate (kapp). Plotting of kapp values as a function of the substrate concentration was fitted to the Michaelis–Menten equation:Vmax is the apparent maximal reaction rate (expressed as pmols x min−1x [E]0) and Km is the apparent Michaelis constant for substrate binding. Each kapp value was derived from the interpolation of 4 different time points. Data were analysed by the program GraphPad Prism 5.0 using Equation  are shown in Supplementary Figure S2.For the determination of the quation:\documentquation:\document1 , again consistent with cleavage starting at the 5′ side of the ssRNA loop. PA does not have any known sequence specificity, thus it can in principle cut anywhere on the RNA substrate. The data shown on Figure Time course experiments were performed with PA on substrates s Scheme : substras Figure  and D. T1 and 2, in the presence of Mg2+. As shown in Figure 1 PA-X generated multiple cleavage products. The earliest products (2 min) corresponded to cleavage starting within the internal bubble (+8) down to the first nucleotides at the 5′ end (+2). At later time points (10 min), products corresponding to cleavage within the U-U-U-U loop (position +18) and to the 3′ side of the internal bubble (positions +28 to +31), started to appear. On the other hand, on substrate 2, PA-X showed a distributive pattern with the generation of a ladder of products similarly to PA 40 ssRNA (substrate 6). As shown in Figure 11, Scheme 7, Scheme In order to further investigate the different substrate specificities of PA and PA-X, a set of different RNA substrates was used Scheme . First, 8, Scheme 8 was placed at the 3′ end. As shown in Figure In order to better understand the differences in cleavage specificity of PA and PA-X, a linear RNA 56mer substrate with a central 18 nt ds region  and 5′ poly (U) tails, thus bearing shorter ssRNA overhangs . We next determined the kinetic parameters Km and Vmax and the catalytic efficiency Vmax/Km, for all four proteins. As reported in Table 1 showed a 33-fold lower activity , with respect to PA-X full length and a 6.7-fold reduction with respect to PAcd. The PAcd itself showed lower activity with respect to PA full length (4.7-fold) and PA-X (3.3-fold). Similarly, on substrate 6, the preferred substrate for PA-X, the catalytic efficiency of PAXΔC20 was 7-fold lower than the one of PA-X (Table Km for the RNA substrates (lower affinity) and a decreased Vmax. Collectively, these results suggest that the C-ter domain of PA and PA-X, albeit different, increased the catalytic activity of the enzyme with respect to the core N-terminal catalytic domain alone. Surprisingly, however, the natural 20 aa deletion present in PAXΔC20 showed a severe deficiency in its endonucleolytic activity.Since PA, PA-X and PAXΔC20, all share the same catalytic domain, in order to understand whether their different C-ter portions may modulate the enzymatic activity, we compared their catalytic activities with the isolated PA catalytic domain (PAcd), representing the first 191 aa in common among all proteins. As shown in Figure in vivo.The endonucleolytic activity of the PA subunit of the RdRP of IAVs, removes the 5′-capped terminus of cellular pre-mRNAs, generating 5′-capped RNA oligonucleotides of 10–13 nt, which are used to prime the synthesis of viral mRNAs . Whetherin vitro transcription assays, using leader RNAs with different degrees of complementarity to the vRNA 3′ end.The results presented here, lend further mechanistic support to these observations. In our study, PA was found to be able to preferentially cut ssRNA regions embedded in dsRNA molecules, such as bubbles or loops. These secondary structures are likely to be formed through imperfect matching between the leader RNA sequence and the vRNA template. In addition, when assessed on linear dsRNA templates bearing ssRNA overhangs, PA cleavage products were consistent with the enzyme cutting the ssRNA region about 10–12 nt downstream from the end of the RNA strand. This is consistent with the results mentioned above and may suggest that the size of the cleavage product corresponds to the size of the ssRNA region that is covered by the enzyme once bound to the substrate. Based on our findings, it is possible to hypothesize that, when the leader cellular RNA, upon binding to PB2 through its 5′ cap, is brought in contact with PA, the alignment between the leader RNA and the 3′ end of vRNA is stabilized through random base-pairing. Cleavage by PA may than occur in correspondence of ds-to-ssRNA junction, i.e. within bubbles formed by the presence of non-complementary regions between the leader RNA and the vRNA. The size of the cleaved product may be determined by the position of the PA active site, which is located 10–12 nt downstream the 5′ capped end. That the size of the cleaved products may depend upon the structural arrangement of the PA subunit, is also suggested by the observation that other cap-snatching viruses show a different cleavage pattern. For example, the Tomato Spotted Wilt Virus has preference for base pairing regions 16 nt downstream the 5′ cap . This hyRecently, it has been shown that a second protein, PA-X, is translated from the IAV genomic segment 3 encoding PA, through a ribosomal frameshifting event . Since PThe PA-X protein is highly conserved among IAVs. However, a natural variant has been found in a subset of IAVs lineages, bearing a nonsense mutation at codon 42, resulting in a truncated PA-X protein lacking the last 20 aa ,13. ThisIn this work, we provide, to the best of our knowledge for the first time, direct evidence that the recombinant PAXΔC20 variant protein has severely reduced enzymatic activity, when compared to PA-X and PA. Our results suggest that the last 20 aa at the C-ter of PA-X, which are not part of the active site, are important for the activity. Crystal structures of PA, both alone or in complex with the other subunits and RNA, have shown that the PA C-ter region (aa 631–716) folds into three α-helices, which closely interact to form part of the PB1 binding site ,22–23. IIn a previous work, the effects of PA-X in modulating gene expression have been compared using an A/Brevig Mission/H1N1/1918 genetic background. The authors showed that lack of PA-X expression increased the overall pathogenicity of H1N1/1918 in mice, leading to differential expression (both up- and down-regulation) of > 5000 genes . Two recDue to its essential role in viral replication, the endonuclease activity of PA is being considered as an attractive target for antiviral chemotherapy. Here we show that both PA and PA-X are inhibited by the small molecule DBPA. Besides demonstrating the possibility of pharmacologically modulating PA-X activity, these results also indicate that it would be important to include PA-X testing in the drug development process, when attempting to develop selective PA inhibitors.


We found that both ZBP1 and HuD bind the zipcode element in rat β-actin mRNA's 3′ UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3′ end of a 5′-ACACCC-3′ motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the β-actin zipcode, but they recognize different features of this target sequence.Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated  The subcellular localization of RNAs has emerged as an important molecular mechanism that is evolutionarily conserved for restricting certain transcripts and proteins to a specific subcellular locale. Current evidence suggests that axonal mRNA transport and local protein synthesis are required for new growth cone formation, retrograde signaling, axon guidance, and elongation ,2. Elucicis-elements mostly present within the untranslated regions (UTRs) of the mRNAs, which are specifically recognized by trans-acting RNA-binding proteins (RBPs), and assembled into large ribonucleoprotein (RNP) complexes (cis-element within the 3′ UTR and its corresponding zipcode binding protein 1 (ZBP1)  –6. The A1 (ZBP1) –9. Transin vitro and in vivo experiments. However, few studies have attempted to identify and determine the precise binding preferences of different RBPs for the same target RNA.Over the last decade, several computational approaches have been developed to predict RBP-RNA interactions ,11. HoweRecent advances in cross-linking immunoprecipitation (CLIP)-based technologies have enabled the study of RBP–RNA interactions on a genomic scale at high resolution –15. Althcis-element both specifically and directly. On the basis of our results, we demonstrate a direct comparison for substrate recognition and binding by the HuD and ZBP1 proteins in an apparently mutually exclusive manner.Recent findings indicate that both ZBP1 and HuD can associate with β-actin mRNA and direct its transport, stability, and translation ,19–21. AAll animal protocols were approved by the Institutional Animal Care and Use Committee, and the experiments were conducted under the IACUC at Alfred I. duPont Hospital for Children.l-lysine (Sigma) and laminin (Millipore) at 37°C, 5% CO2.L2–6 DRGs were dissociated with 50 U collagenase type XI (Sigma) for 25 min at 37°C and then triturated with a fire-polished Pasteur pipette. Dissociated DRG neurons were suspended in ‘nucleofector solution’ (Lonza), and 5–7 μg plasmid was electroporated using an AMAXA 4D-Nucleofector system (Lonza). Complete culture medium in DMEM:F12 (Life Technologies), 1x N1 supplement (Sigma), 5% horse serum, 5% fetal bovine serum (Hyclone), and 10 μM cytosine arabinoside (Sigma) was directly added into the transfection cuvette, and DRG neurons were plated on coverslips coated with poly-Escherichia coli BL 21 (NEB) transformed with pGEX-GST-HuD plasmid was diluted 1:50 in LB medium. At an OD600 of 0.4, the culture was induced with isoproryl-β-D-thiogalactopyranoside  (Life Technologies) at 37°C. Four hours later, the cells were spun down and resuspended with purification buffer . The cells were lysed by the addition of lysozyme (0.2 mg/ml) and sonication. The lysate was incubated at 4°C for 15 min with 1% Triton X-100 (v/v) and 1% sarkosyl (w/v) for solubilisation. It was then centrifuged 16 000 g for 15 min, and the resulting supernatant was collected and passed through 45 μm filter membrane. GST-Sepharose 4B beads  were used to purify GST-HuD protein. Purified GST protein was eluted with 20 mM glutathione in 50 mM Tris–HCl pH 8.0, desalted using centrifugal filter unit.Coding region of rat HuD protein was cloned into pGEX-2T GST construct , and rat ZBP1 protein sequence was cloned into the same construct and GST was replaced with 6X His for further purification. An overnight culture of E. coli BL 21 transformed with pGEX-His-ZBP1 plasmid was induced with 0.1 mM of IPTG at 27°C for 6 h. After spinning down, cell pellet was re-suspended with buffer . Ni-NTA beads (Life Technologies) were used for purification. Purified His-ZBP1 protein was eluted with 250 mM imidazole.His-ZBP1 was purified as described above with slight modification. In brief, Bradford and BCA assays (Bio-Rad) were used for quantification of GST-HuD and His-ZBP1 proteins, respectively. Concentration of the purified proteins was confirmed by comparison with bovine serum albumin (BSA) standards in acrylamide gel stained with Coomassie brilliant blue. Purity after purification was evaluated by Coomassie brilliant blue staining, and the sample with higher than 95% purity was used for binding assays.2, 40 mM KCl, 1 mM DTT, 400 U of RNaseOUT™ and 5% glycerol) at 4°C for 30 min. After washing, beads were incubated in TRIzol (Life Technologies) and extracted by phenol–chloroform to isolate RNA. RNA was precipitated with ethanol using a glycogen carrier.Purified GST-HuD and His-ZBP1 proteins were probed with Dynabead Protein G (Life Technologies) conjugated with anti-GST and anti-His antibody (Life technologies). Beads conjugated with mouse IgG were used to see the background binding. Beads were washed with binding buffer (Promega) and incubated with 1 μg of total RNA isolated from rat dorsal root ganglion (DRG) in binding buffer  as described in the technical manual. The transcribed RNAs were biotinylated on their 3′ end using RNA 3′ End Biotinylation Kit (Thermo Scientific Pierce). Ribogreen assay (Life Technologies) was used to quantify concentration of RNA, and dot blot was performed to confirm the biotinylation efficiency. The 54 nucleotides zipcode motifs from rat and mouse mRNA and zipcode mutants were customized from IDT (Integrated DNA Technologies). For binding assay, RNAs were biotinylated at both ends. In some experiments, an excess of cold zipcode RNA was used to confirm the specificity of the assays. A reaction mixture (20 μl) containing 20 fmol of RNA and 3 pmol of purified protein was incubated in a binding buffer  at room temperature for 20 min. After incubation, the binding reaction was resolved on a native 6% polyacrylamide gel in 0.5× TBE and transferred to a nylon membrane. Band shifts were detected using the Chemiluminescent Nucleic Acid Detection Module (Pierce).The pcDNA3 plasmid (Life Technologies) containing the full-length 3′ UTR of rat β-actin mRNA was linearized with XhoI restriction endonuclease and transcribed Kd, apparent) between the wild-type and mutant RNA for ZBP1 was obtained using three independent REMSA experiments. The fraction of bound RNA was plotted graphically versus protein concentration, and the binding capability of ZBP1 for the wild-type RNA and mutant was compared. Nonlinear regression fits of the data revealed an apparent dissociation constant equal to 28.1 nM for wild-type RNA, statistically different from the Kd, apparent = 315.4 nM for the mutant RNA (P < 0.0001).The apparent dissociation constant  and confirmed again with Vienna Fold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). In case RNA shows more than one prediction, the structure with the lowest ΔG was considered as main structure.Secondary structure of RNA was predicted using web-based software, mFold (® RNase III (NEB) was used for RNA cleavage assays. The enzyme cleaves dsRNA into short dsRNA fragments (18–25 bp) in a manganese-containing reaction buffer. Cleavage reactions were performed in 20 μl volumes containing 10 fmol of biotin-labelled wild-type or mutant zipcode RNAs. 0.4 or 4 units of RNase III were added to the reaction and incubated at 37°C for 20 min. EDTA was added to stop the reaction and immediately loaded onto 6% denature acryl amide gel  after mixing with 2× denaturing loading buffer . After running, each gel was transferred onto positively charged nylon membrane and developed as described above in REMSA method.ShortCut−/−HuD mouse (HuD KO) according to Bird et al.  and processed further for quantitative PCR (qPCR) using an Applied Biosystems 7300 Real-Time PCR System. β-Actin mRNA signals were normalized to GAPDH mRNA using the comparative threshold (Ct) methods and expressed as fold change relative to t = 0 min from the 2Ct)−. The plasmid was linearized with restriction endonuclease and transcribed in vitro using Riboprobe® In Vitro Transcription Systems (Promega) to generate cRNA probe for in situ hybridization as described in the technical manual. Antisense probe for eGFP reporter was transcribed from SP6 promoter and sense probe was transcribed from T7 promoter. Probes were chemically labelled with digoxigenin succinamide ester (Roche) during in vitro transcription. cRNAs were then alkaline hydrolysed to 100–150 nt length. All probes were stored at −80ºC until used.The wild-type and mutants of zipcode sequence were incorporated into the E. coli tRNA, and 0.5× Denhardt's solution containing 5 ng cRNA per coverslip. Coverslips were then washed twice for 20 min in 50% formamide/2× SSC at 37ºC, followed by three washes in 1× SSC for 10 min each on a rotary shaker.FISH for DRG cultures was similar to previously described methods . All steFor subsequent IF, samples were blocked in 50 mM Tris, 150 mM NaCl, 2% BSA, and 2% fetal bovine serum (‘IF buffer’) for 1 h. After blocking, samples were incubated for 1 h in the following primary antibodies in IF buffer: chicken anti-neurofilament (NF) H , chicken anti-peripherin , and sheep anti-digoxigenin . Samples were washed in TBS buffer three times and then incubated with Alexa633-conjugated anti-chicken IgG antibody  and Alexa488-conjugated anti-sheep IgG antibody  in IF buffer for 1 h. Coverslips were mounted using PVA-DABCO (Sigma) anti-fading mounting medium and visualized with Leica DMRXA2 epifluorescent microscope. All images were matched for acquisition parameters.Graphpad Prism 6 software package (GraphPad) was used for statistical analyses. Non-linear regression analyses were used for the competitive binding experiments, the apparent dissociation constant between the wild-type, mutant, and chicken RNAs for ZBP1, and β-actin decay experiments.via direct or indirect interaction. Furthermore, there have been relatively few studies for a direct comparison of the binding preference of different proteins to the same RNA target. We hypothesized that differential binding preference of different RBPs toward the same target RNA would provide an additional mechanism to increase specificity of mRNA localization into distal processes of neurons. To ascertain that the precipitation of β-actin mRNA with ZBP1 or HuD protein that we have previously shown is in fact dependent on direct protein–RNA interaction, rather than through indirect protein-protein interactions, both full ZBP1 and HuD were expressed and purified . Briefly, whole cell lysate of E. coli BL15 strain expressing either GST-tagged HuD (HuDGST) or His-tagged ZBP1 (ZBP1His) was incubated with Glutathione-Sepharose beads or Ni-nitrilotriacetic acid (Ni-NTA) beads, respectively, followed by removal of nonspecific proteins with extensive washing. After eluting, the proteins were concentrated, quantified, and analyzed by SDS-PAGE and western blotting .Our recent studies using quantitative real-time PCR (qRT-PCR) showed that β-actin and GAP-43 RNAs were detected both in the HuD immunoprecipitate and ZBP1 immunoprecipitate with relatively different levels of enrichment, suggesting a different affinity of these proteins for different mRNAs . Howevertrans-acting factors that specifically recognizes a 54-nt sequence in the 3′ UTR of chicken β-actin mRNA, called the zipcode that is required and sufficient for its subcellular localization and translational regulation (in vitro then biotinylated. The RNA electrophoretic mobility shift assay (REMSA) was conducted to resolve the biotin-labeled 3′ UTR in complex with purified HuD and compared with ZBP1-β-actin 3′ UTR complex .ZBP1 is one of the best-studied gulation . Althouggulation –21, we ky Figure . Howevercis-acting elements are in fact quite varied in sequence .To directly test if HuD binds the zipcode element, we analyzed binding of HuD to the RNA using the purified proteins and synthetic wild-type zipcode RNA of the rat β-actin 3′ UTR. As shown in Figure G fragment from the tau mRNA's 3′ UTR as a positive control. The 600-nt G fragment has been shown to contain direct binding sites for both ZBP1 and HuD  or HuD (closed circle) Figure . The amo and HuD ,32–33. Tcis localization sequence for β-actin mRNA in chicken fibroblasts via binding of ZBP1 protein . In particular, we found that a 5′-ACACCC-3′ sequence (shaded in red) and U-rich sequence UU(uu)UCU(u)U (shaded in blue), juxtaposed to 3′ end of the ACACCC within the zipcode, were highly conserved for all mammals examined, suggesting that these regions within the zipcode are functionally important. The ACACCC motif was originally discovered as a minimal  protein ,34–35. T protein ,32,36–38To search for putative secondary structure(s), the zipcode elements of rat and chicken β-actin 3′ UTRs were further analyzed with several web-server-based, RNA folding prediction algorithms such as Mfold and CentroidFold Figure . The ratTo investigate the recognition and binding preferences of the zipcode RNA by the two different RNA-binding proteins, HuD and ZBP1, we tested a series of sequence-mutated zipcode RNAs on their association with the purified HuD and ZBP1 proteins. First, in an effort to determine whether the primary sequence is sufficient for both HuD and ZBP1 binding, the conserved ACACCC or UUUCUUU motif of the rat zipcode element was simply deleted from the zipcode sequence, and REMSA experiments were carried out with purified proteins. Given the importance of ACACCC motif for ZBP1 recognition and binding ,35, we eTo determine if the integrity of the predicted secondary structure of the zipcode element is functionally important for both HuD or ZBP1 binding, the ACACCC motif or U-rich motif-deletion mutants of the zipcode RNAs were further analyzed by using the RNA folding prediction Mfold program. As shown in Figure All of the above data indicated that the specific binding determinant(s) on the zipcode RNA for ZBP1 and HuD proteins seemed to be specific feature(s) with the secondary structure. To ascertain that the proposed secondary structure of the zipcode RNA displaying similar features with that of the wild type zipcode is sufficient for ZBP1 and HuD binding for subcellular localization, a mutant zipcode RNA was carefully designed, in which the conserved U-rich 5′-UUUCUUU-3′ sequence was replaced with 5′-CCUCCCU-3′, but the predicted structure was not disturbed Figure . These rAAMTCG zipcode), in which only the structure was potentially disrupted without changing the ACACCC and U-rich motifs . In contrast, purified HuD protein showed no apparent changes in the shifted gel mobility of the complex with the mutant RNA, compared to that with the wild-type zipcode  for ‘unmasked’ β-actin mRNA reported in dendrites of cultured hippocampal neurons  was used to directly visualize reporter mRNAs with cRNA probe specific for the reporter mRNA. We first checked to see if all of these reporter mRNAs are expressed in the transfected DRG neurons by focusing on GFP mRNA FISH signals in cell bodies . DRG neurons transfected with this construct showed only weak axonal mRNA signals compared with neurons expressing wild-type zipcode, indicating that HuD binding alone is not efficient for targeting reporter mRNA into axons. These observations were also consistent with the reduced Kper se in the rat β-actin mRNA's 3′ UTR is not sufficient for binding of ZBP1 to target its RNA to the final destination, and that ZBP1 and HuD recognize the same target zipcode RNA in a fundamentally different way with differential relative preference.We present here that HuD primarily recognizes and binds to the primary sequence feature in the zipcode of rat β-actin mRNA, whereas the ZBP1 preferentially requires a proper spatial arrangement of the zipcode for binding, as previously determined ,49. Furtvia AREs in the 3′ UTR and forms an mRNP complex with the survival of motor neuron (SMN) protein. HuD-SMN complex then transports the associated cpg15 mRNA to axonal processes of spinal motor neuron, as well as regulates the local stability or translation, or both. SMN can also bind hnRNP-Q/R protein, which localizes β-actin mRNA to axonal growth cones of motoneurons  mRNA interacts with HuD oneurons –52. Althoneurons .in vitro was not affected by the absence of ZBP2 but drastically hampered by HuD , is known to participate in destabilizing its target mRNAs ,54–55. SD Figure , indicatD Figure , implicacis-element within the 3′ UTR , KSRP, tristetraprolin (TTP), T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1-related protein (TIAR) has been implicated in mRNA degradation and/or translational repression. However, the ELAV/Hu proteins positively regulate the stability and translation of their target mRNAs –58. Conse 3′ UTR ,35,59–60e 3′ UTR ,61, we ain vitro ,63. Therin vitro ,65, it icis-element in its 3′ UTR (cis-element as well as a combination of different zipcodes mediating distinct steps in localization (Spatial and temporal restriction of gene expression to specific subcellular domains, especially of neurons, is critical to achieve autonomous response to external stimuli. Increasing evidence demonstrates that specific interactions between RNA localization elements and cellular factors play an essential role in cytoplasmic sorting of mRNAs to their destination –68. Thess 3′ UTR . We prevs 3′ UTR . It is alization . Complextrans-acting factors including not only RBPs but also small non-coding regulatory miRNAs (trans-acting factors are particularly important for highly polarized neurons, in which specific neuronal mRNAs have to be stabilized to survive and translationally repressed during their transport. To this end, the interactions of mRNAs with RBPs and/or miRNAs involve the functional cooperation between different trans-factors that can work in collaboration or exert opposing effects on target mRNAs. As detailed in Supplementary Figure S6, ZBP1 and HuD might compete for binding to a single cis-element but co-reside on the same mRNA as it is transported from its site of synthesis in the nucleus to its final destination.In addition to the localization sequence elements of mRNAs, the stability, transport, and translation of an mRNA are determined, to a large extent, by dynamically interacting y miRNAs –44,71–73in vitro, we employed RNA electrophoretic mobility shift assay with purified proteins and multiple synthetic RNA substrates whose structures were predicted with RNA folding prediction algorithms. Although our understanding of how RBPs recognize/bind their target RNAs is still very limited and the capability to predict which RBP binds which RNA is not in any way plausible, relatively recent biochemical studies by Singer and coworkers showed that the spatial orientation of ZBP1 KH domains 3 and 4 at opposing ends of ZBP1 leads the chicken zipcode RNA to loop around ZBP1 to an anti-parallel orientation (To obtain further insight into the interaction modes of HuD and ZBP1 with the same zipcode RNA entation ,49. Whilentation –76. In tentation ,78. In centation ,79. ThusTaken together, these results provide an experimental basis suggesting that the interplay of multiple RBPs for the target RNAs can play unique roles, independently or cooperatively, in mRNA metabolism including stability, localization, and translation, as well as allow effective redistribution of the mRNAs in response to local stimuli. Of course, understanding their interaction relating to mRNA metabolism for normal biological properties and, most importantly, for an eventual disease context, will require future analyses.Supplementary Data are available at NAR Online.


Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein. Mitochondria are involved in numerous cellular functions, including many biosynthetic pathways, calcium signaling, apoptosis, innate immune responses, and production of ATP through oxidative phosphorylation (OXPHOS). Although most mitochondrial proteins are encoded in the nucleus and imported into the mitochondria, this organelle also contains its own genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are essential for OXPHOS, and also includes genes for two rRNAs and the 22 tRNAs that are needed for protein translation on mitochondrial ribosomes. The mtDNA exists in a nucleoprotein complex known as the mitochondrial nucleoid.Mitochondrial Transcription Factor A (mt-TFA/ TFAM) is an HMG-box protein that was initially identified as a mtDNA transcription factor over 25 years ago in the Clayton lab . Its rolIn addition to RNA, mitochondrial nucleoids are also associated with numerous ribosomal proteins and other factors involved in protein synthesis ,12. TFAMThe mouse TFAM cDNA  was amplified using the PCR primers 5’-agccatatgtccagcatgggcagctat-3’ and 5’-gctggatccttagtgttcggagatgtc-3’, and cloned into the NdeI and BamHI sites of pET28a . The resulting fusion protein excludes the N-terminal 32 amino acid mitochondrial localization sequence of TFAM, which is replaced with 23 amino acids containing a 6X HIS tag and the thrombin cleavage site. The clone was validated by nucleotide sequencing, and was used to transform the E. coli BL21(DE3) for expression using Overnight Express™ auto induction media (EMD4 Biosciences). Bacteria from 500 ml cultures were lysed using B-PER bacterial protein extraction reagent and purified using B-PER nickel-chelated columns (Thermo Scientific). In some experiments, the non-native 6X HIS leader was cleaved using 5 U of thrombin/mg of HIS-tagged TFAM. The cleaved peptide was removed by passage over nickel-chelated columns as above, and thrombin was removed using a HiTrap Benzamidine FF column . Purified protein was stored at -80°C in 50 mM Tris, 1 mM EDTA, 150 mM NaCl, 10% (v/v) glycerol.Several commercially available antibodies against TFAM failed to perform in immunoprecipitation experiments. Therefore, a custom polyclonal antibody was generated in rabbits against bacterially expressed, HIS-tag purified, full-length mouse TFAM protein . The antibody was validated by Western blot analysis of cell lysates and by immunofluorescence imaging where the antibody stained mitochondrial nucleoids in a manner identical to an anti-DNA antibody . Western2 was also added. Reactions contained 20 fmoles of nucleic acid substrate and varying amounts of TFAM. Binding was allowed to occur for 30 min at 30°C prior to gel electrophoresis in 6% PAGE, 0.5X TBE gels (Invitrogen). Nucleic acids were transferred to positively charged nylon membrane (Roche), and crosslinked with UV light. Biotinylated substrates were detected using stabilized streptavidin-HRP/Luminol reagents as components of the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific). Quantification of free and bound nucleic acids was performed as above using the ImageQuant LAS400. Estimations of the apparent Kd values from EMSA data were determined from Hill plots where the x-intercept is the log (Kd). Binding curves were plotted and linear regression analysis was done using GraphPad Prism. Several test substrates yielded two species of shifted bands, which likely represent multiple TFAM binding sites on these substrates 20Single stranded RNAs; biotinlylated poly [rArC]Internal loop dsRNA substrates; RNA oligo .Stem-loop RNA substrate; RNA 5’-biotin-rArGrU rCrUrG rGrArC rArCrG rUrArC rUrUrU rUrUrG rUrArC rGrUrG rUrCrC rArGrA rCrU-3’, which self anneals, creating a 15 nt stem with a 5 nt bubble at the hairpin.4-way junction oligos; DNA versions shown here. Corresponding RNA oligonucleotide set was also synthesized.b strand, 5’-biotin-TCCGTCCTAGCAAGGGGCTGCTACCGGAAG-3’;h strand, 5’-CTTCCGGTAGCAGCCTGAGCGGTGGTTGAA-3’;r strand, 5’-TTCAACCACCGCTCAACTCAACTGCAGTCT-3’;x strand, 5’-AGACTGCAGTTGAGTCCTTGCTAGGACGGA-3’The FluoroTect™ GreenLys is a charged E. coli lysine tRNA labeled with a BODIPY fluorophore and was purchased from Promega. EMSA reactions with this substrate contained 316 fmoles of nucleic acid substrate and varying amounts of TFAM. Binding conditions were as described above. Imaging and analysis of gel shifted bands was done with an ImageQuant LAS400 Imager and ImageQuant TL  using the fluorescein LED and filter for the detection of BODIPY.GeneSwitch 3T3 cells (Invitrogen) were cultured in high glucose DMEM supplemented with 10% (v/v) newborn calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate and [50 μg/ml] hygromycin-B (Invitrogen). For confocal microscopy, cells were grown in Lab-Tek II chambers with #1.5 borosilicate coverglass bottoms (Nunc) coated with 15 μg/ml human fibronectin (Millipore). Immunofluorescence was performed by fixing cells for 30 min at 37°C with 4% (w/v) paraformaldehyde in 0.1 M phosphate, pH7.4, permeabilized with 0.25% (w/v) triton X-100 in PBS. Where appropriate, samples were treated with either or both [0.1mg/ml] DNase-free RNase or [50 U/ml] RNase-free DNase (Roche) for 90 min at 37°C, blocked in 5% (v/v) goat serum and incubated with affinity purified anti-TFAM polyclonal antibody (Primm) diluted 1:3000 in 0.25% Triton X-100 in PBS overnight at 4°C. Cells were labeled with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Invitrogen) diluted 1:4000 in 0.25% (w/v) Triton X-100 in PBS for 1 h at room temperature. DNA was labeled with anti-single-stranded DNA (anti-ssDNA) monoclonal antibody (clone BV16-19 [Millipore]) diluted 1:4000 as above, and secondarily with an Alexa Fluor 568 goat anti-mouse IgG secondary antibody (Invitrogen) diluted 1:3,000 as above.Confocal imaging was performed on a Zeiss LSM 510 META microscope equipped with a 100x/1.4NA Plan-Apochromat objective, and the 488 line of a 30 mW multi-line gas argon laser. All images are 1024x1024, 12-bit z-stack acquisitions with 0.8 μm steps. Fluorescence per cell was quantified using Volocity (Perkin Elmer) to find objects by intensity and clipping objects to ROI. Microsoft Excel was used for statistical analysis.2 flasks to 80% confluency, washed with PBS and crosslinked with 1% (w/v) formaldehyde for 10 min. Crosslinking was quenched with excess glycine, and cells were again washed with PBS prior to harvesting by scraping and centrifugation. Cell pellets were frozen and stored at -80°C. Lysates were prepared by adding 0.6 ml of 50 mM Tris-HCl, pH 8.0, 1% (w/v) SDS, 10 mM EDTA, 50 U/ml SUPERase-In (Life Technologies), and protease inhibitors  to each T-150cm2 derived pellet. Cell suspensions were sonicated in a Covaris TC13 tube using a Covaris S2 with a duty cycle of 5%, an intensity setting of 4, and 200 cycles per burst. This was run for seven repetitions of 2 min intervals. Lysates were diluted with 9 volumes of IP buffer . For each sample, 3 ml of lysate was treated with 6 μg of anti- TFAM polyclonal antibody (see above) and immune complexes were allowed to form at 4°C for 3 h with rotation. Immune complexes were immobilized by adding 50 μl of Dynabeads® Protein G (Life Technologies), allowed to bind for 2 h at 4°C, and collected magnetically. Beads were sequentially washed once each with 1 ml of a low-salt buffer  Triton X-100, 0.1% (w/v) SDS), a high-salt buffer  Triton X-100, 0.1% (w/v) SDS), LiCl buffer  NP40, 1% (w/v) deoxycholate) and TE, pH 8.0. Immune complexes were then eluted with 100 mM sodium bicarbonate, 1% (w/v) SDS, 50 U/ml SUPERase-In. For RNA analysis, samples were treated with TURBO™ DNase (Life Technologies). Samples were then treated with proteinase K and crosslinks were reversed by adding 20 mM NaCl and heating at 65°C for 18 hr. Nucleic acids were precipitated in isopropanol, resuspended in water and stored at -20°C.Mouse 3T3Sw cells were grown in T-150 cmRNA samples were treated again with TURBO™ DNase (Life Technologies), followed by inactivation. cDNA synthesis was done using Superscript III RT (Life Technologies) as outlined by the manufacturer. Reverse transcriptase was omitted in parallel samples to control for contaminating DNA. The primers used for cDNA synthesis and amplification are listed in TFAM binding kinetics to a dsDNA LSP sequence, a 4-way DNA junction, a 4-way RNA junction and to native mt tRNAs were obtained using a Biacore T100  in a manner similar to that used previously . BiotinyMitochondrial tRNAs were prepared from mouse liver tissue as described . Livers TFAM -bound tRNA was isolated using RNA immunoprecipitation as outlined above. Total cellular RNA was isolated from cultured 3T3sw cells using the AllPrep DNA/RNA mini kit (Qiagen). RNA from each was circularized with T4 RNA ligase 1, followed by RT-PCR using the Superscript III first-strand synthesis kit (LifeTechnologies) per the manufacturers instructions. Primers tRNA V RT FS (5’-gtgtaggccagatgctttaata-3’) and tRNA V F (5’-ccagaagatttcatgaccaatg-3’) were used to amplify a 69-nt fragment, which was subcloned using the TOPO-TA cloning system (Life Technologies). Individual clones were isolated and the plasmids were sequenced using the M13 forward primer.Kd of 6 nM. This value is similar to that measured by others, thus validating the assay [Kd of 100 nM for this substrate. With regard to RNA substrates, we demonstrate that TFAM is unable to bind to linear RNA:DNA hybrids, double-stranded RNA (dsRNA), or the single-stranded RNA (ssRNA) substrates poly (rArC) or poly(rU)  . The mithe assay . To measpoly(rU) . Thus, tKd of 2.04 μM, which is over 300-fold higher than the apparent Kd for the LSP dsDNA. TFAM has also been shown to bind to DNA 4-way junctions, which are typical of HMG-box proteins [Kd of 63 nM. We then looked for the ability of TFAM to bind to RNA- and RNA:DNA-containing 4-way junction substrates and were able to qualitatively demonstrate binding. Several of the 4-way junction substrates yielded two binding isoforms that fill sequentially with increasing TFAM. The Hill coefficients for the 4-way substrates were 1.6–3, indicating cooperative binding of TFAM. This is consistent with previous studies in which TFAM binding to specific and nonspecific substrates yielded Hill coefficients of 1.5–2. [Kd. Binding affinity estimates indicate that the RNA-only 4-way junction has an apparent Kd of 270 nM (Kd that is similar (~299 nM) to the all-RNA 4-way junction. However, when the 4-way structure contains some RNA:DNA, some RNA:RNA, and some DNA:DNA paired structure, the apparent TFAM affinity is greater (Kd ~63 nM), and is identical to the all-DNA 4-way junction (Kd ~63 nM). This likely indicates that TFAM is preferentially binding to the dsDNA arm of this substrate when given a mixed arrangement around the junction.RNA most often forms structures that are not simply linear. We therefore assayed the capacity of TFAM to bind more complex substrates. EMSA data show that TFAM is unable to bind to a dsRNA hairpin loop, which is a common secondary structure in RNA . Howeverproteins . We havef 1.5–2. . We havef 270 nM . The appUUR, which was below detection. The relative ability to detect tRNAs purified by TFAM RIP are ranked and displayed in UUR was undetectable in the TFAM immunoprecipitates, although our cellular assays indicated that this tRNA was more available than 9 of the other 19 tRNAs detected. Thus, although total cellular tRNA abundance may partially influence the amount that is bound to TFAM, it is not the primary factor. We also looked for TFAM binding correlations related to tRNA structural features without success. The determinants influencing the ability of TFAM to bind to a particular tRNA or our ability to detect those tRNAs is therefore not yet clear.Since the EMSA binding data indicated that TFAM binds to complex RNA structures, we sought to identify TFAM-bound mitochondrial RNAs by using RNA immunoprecipitation (RIP) . We usedTranscription of the circular mtDNA proceeds from strand-specific promoters in opposite directions, creating both sense and antisense transcripts from both strands. We looked for the antisense RNAs corresponding to the tRNA regions and found that they were below detection in TFAM RIP samples. Therefore only bona fide tRNAs are detected in our TFAM immunoprecipitations. Finally, we assayed for cytoplasmic  tRNA Arg, tRNA Pro, and tRNA Val to determine if TFAM was specifically binding mitochondrial tRNAs. We were unable to detect these cytoplasmic tRNAs in the TFAM RIP samples (data not shown), confirming our ability to cross link the TFAM to mitochondrial substrates in our immunoprecipitations.Given these in vitro and in vivo crosslinking data, only tRNA or RNA containing cloverleaf-like junctions would be expected to contribute to TFAM binding within the mitochondria or their nucleoids. We localized and quantified the relative amounts of DNA- and RNA-bound TFAM using cellular immunofluorescence. Kd of 0.27 nM and 3.75 nM) are greater than those of the RNA substrates . The difference in these affinities is largely explained by the dissociate rate constants (kd). TFAM is more easily dissociated from the RNA substrates, and remains more steadily bound to the DNA substrates. Therefore, TFAM binding to the RNA 4-way junction and to mitochondrial tRNAs is more transient, relative to dsDNA and DNA 4-way junctions.The EMSA derived binding affinities are complicated by multiple binding events see , impreciAGY, and Pro were all fully processed at their 5’-ends. Similarly, mt-tRNAs for Phe, Val, and Met were fully processed at their 3’-ends. All of these tRNAs were only detectable as processed versions, using primers within the boundaries of normal 5’- and 3’-ends.Since mtDNA is packaged by TFAM, we postulated that it might bind tRNA transcripts immediately after transcription. TFAM binding of nascent RNA might indicate involvement in early steps of tRNA maturation. We used RT-PCR to determine if TFAM binds tRNAs that have yet to undergo end-processing by looking for longer versions of these RNAs. We then employed RNA circularization  to deterKd of 20.4 nM. Thus TFAM is capable of binding this aminoacylated tRNA with an affinity which is similar and perhaps even higher than what we measured for total mitochondrial tRNAs using SPR . sing SPR .In this work, we have characterized the ability of TFAM to bind to various RNA containing substrates in order to elucidate the potential roles of this protein in both nucleoid structure and mitochondrial RNA function.TFAM is the predominant architectural protein of the nucleoid, where it is involved in both transcription and maintenance of mtDNA. This HMG-box protein binds with high affinity to mtDNA promoters, bending and opening the DNA for initiation of transcription. Apart from the promoter regions, TFAM also binds to mtDNA nonspecifically; condensing it via DNA bending and loop formation ,21. The We have also found that TFAM binds with nanomolar affinity to mitochondrial tRNAs. TFAM RNA immunoprecipitations did not yield mitochondrial mRNAs, antisense mitochondrial tRNAs, or non-mitochondrial tRNAs. This does not exclude the possibility that TFAM may bind some or all of these RNAs in vivo. Mitochondrial tRNAs are over 100-fold more abundant than mitochondrial mRNAs . These aSince TFAM is an abundant DNA binding protein, we first postulated that TFAM might bind to nascent RNA as it is displaced from mtDNA during transcription. Mammalian mitochondrial RNAs are transcribed as polycistronic RNAs originating from two strand-specific promoters on the circular mtDNA. Genes within mtDNA lack introns, and are most often arranged with tRNA genes interspersed between the mRNAs. The excision of tRNAs is coincident with rRNA and mRNA processing . Thus, tKd of TFAM to a mixed population of mitochondrial tRNAs to be about 74 nM, which is equivalent to the equilibrium dissociation constant of bovine mitochondrial EF-Tu-GTP-Phe-tRNA (76 nM)[There have been numerous experimental links made between nucleoid proteins and their involvement in RNA processing, ribosome biogenesis, or translation. In both yeast and humans, the mitochondrial RNA polymerase amino terminal domain binds to proteins involved in RNA processing and translation –42. ProtA (76 nM). The binA (76 nM),47. BogeA (76 nM),49. It hA (76 nM). This tRAlternatively, TFAM may fill a more general need for a mitochondrial tRNA chaperone-like function. Recent evidence indicates that mitochondrial tRNA structure may require stabilization. The C-terminal region of mitochondrial leucyl-tRNA synthetase is able to bind and rescue mutant tRNAs, functioning as a tRNA chaperone which stabilizes the appropriate tRNA structure ,52. ThisFinally, mitochondria play a central role in both apoptosis and the innate immune response  and we cS1 Fig(A) Confocal immunofluorescence image with TFAM antisera and AlexFluor 488 conjugated secondary antibody. (B) Anti-DNA antibody immunofluorescence with Alexa-Fluor 568 conjugated secondary antibody. (C) Merged images from (A) and (B) demonstrating co-localization of TFAM and DNA antisera at mtDNA nucleoids. Scale bar in (A) is 10 μm. (D) TFAM antisera Western analysis showing asterisk-labeled TFAM band at appropriate migration size, and lack of significant cross-reactivity.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Catalytic RNAs, or ribozymes, are regarded as fossils of a prebiotic RNA world that have remained in the genomes of modern organisms. The simplest ribozymes are the small self-cleaving RNAs, like the hammerhead ribozyme, which have been historically considered biological oddities restricted to some RNA pathogens. Recent data, however, indicate that small self-cleaving ribozymes are widespread in genomes, although their functions are still unknown.retrotransposons with hammerhead ribozymes, referred to as retrozymes. These elements contain two long terminal repeats of approximately 350 bp, each harbouring a hammerhead ribozyme that delimitates a variable region of 600–1000 bp with no coding capacity. Retrozymes are actively transcribed, which gives rise to heterogeneous linear and circular RNAs that accumulate differentially depending on the tissue or developmental stage of the plant. Genomic and transcriptomic retrozyme sequences are highly heterogeneous and share almost no sequence homology among species except the hammerhead ribozyme motif and two small conserved domains typical of Ty3-gypsy long terminal repeat retrotransposons. Moreover, we detected the presence of RNAs of both retrozyme polarities, which suggests events of independent RNA-RNA rolling-circle replication and evolution, similarly to that of infectious circular RNAs like viroids and viral satellite RNAs.We reveal that hammerhead ribozyme sequences in plant genomes form part of a new family of small non-autonomous Our work reveals that circular RNAs with hammerhead ribozymes are frequently occurring molecules in plant and, most likely, metazoan transcriptomes, which explains the ubiquity of these genomic ribozymes and suggests a feasible source for the emergence of circular RNA plant pathogens.The online version of this article (doi:10.1186/s13059-016-1002-4) contains supplementary material, which is available to authorized users. The hypothesis of a prebiotic RNA world, where the first living organisms were based on RNA as both the genetic material and as catalyst –3, was sThe occurrence of HHRs has been previously reported in some plant genomes , 17, 22.As previously noticed , we confAnalysis of the elements containing tandem HHR copies showed that ribozymes were embedded in direct repeats of about 300–400 bp, which delimited a central region of about 300–600 bp without any coding potential see Fig.  32]. Th. Th32]. We deduced that transcription of genomic retrozymes followed by self-processing through HHR motifs would result in RNA transposition intermediates  of about 600–1000 nt, depending on the plant species Fig. . These rMet) of the Ty3-gypsy retrotransposons  with RNAMotif revealed up to 30 bona fide type III HHRs, which showed some sequence heterogeneity for the same secondary structure . LTRs were about 330 bp long, and the two HHR self-cleavage sites encompassed a non-coding region of 697–776 bp. At least four genomic retrozyme sequences were detected embedded within 18S rRNA gene sequences, whereas four others were found close (less than 2 kb) to LTR-retrotransposon sequences and the rest were detected within intergenic regions, in a similar way as described for other non-autonomous retroelements like TRIMs and SMARTs , 3734, 3Pospiviroidae UTP, 0.4 U/μl of porcine liver ribonuclease inhibitor (Takara), 20 ng/μl of plasmid DNA and 4 U/μl of T7 (Takara) or T3 (Roche Diagnostics GmbH) RNA polymerases. After incubation at 37 °C for 1–2 h, products were fractionated by polyacrylamide gel electrophoresis (PAGE) in 5 % gels with 8 M urea, and detected by phosphorimaging . For the synthesis of DIG-labelled riboprobes of positive and negative polarity, transcription reactions were carried out in the same conditions as described above, except that radiolabelled UTP was replaced by 0.5 mM digoxigenin-11-UTP (Roche Diagnostics GmbH) and reactions were incubated at 37 °C for 4 h.Retrozyme RNAfragments harbouring one HHR motif were synthesized by in vitro run-off transcription of pBlueScript KS+ plasmids containing the corresponding retrozyme insert previously linearized with 4 dilution in blocking solution; Roche Diagnostics GmbH). The chemiluminiscence produced in the presence of the substrate CDP-Star  was finally visualized in a LAS-3000 Imaging System (Fujifilm).For Northern blot analysis, from 5 up to 100 μg of purified RNA from different plant tissues were examined in 5 % polyacrylamide gels containing 8 M urea and 1× TBE . For double PAGEs, nucleic acids enriched in RNAs of the appropriate retrozyme size were obtained by cutting a section from nondenaturing 5 % polyacrylamide gels. These RNAs were examined in denaturing 5 % polyacrylamide gels containing 8 M urea and 0.25× TBE . After ethidium bromide staining, RNAs were electroblotted to nylon membranes  and UV-fixed with a crosslinker . Prehybridization, hybridization (at 68 °C in 50 % formamide for 16 h) and washing (twice with 0.1× SSC at 68 °C for 15 min) was done following the instructions of the manufacturer . The DIG-labelled probes were detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-digoxigenin-AP Fab fragments, 1:10F. ananassa retrozyme RNAs [GenBank:BATT01039028.1:c14263-13183] was amplified by PCR using primers designed to bind outside the sequence of the retrozyme (Fa92D and Fa92R, Additional file XbaI and BamHI sites of pBlueScript KS+, and the resulting plasmid was XbaI-linearized for run-off transcription with T3 RNA polymerase. The full uncleaved transcript and the retrozyme RNA resulting from self-cleavage at both HHR motifs were fractioned in 5 % polyacrylamide gels containing 8 M urea and 1× TBE, and extracted from gel slices as described above.The genomic retrozyme with the highest sequence homology to most of the cloned Solanum melongena [2, 5 mM DTT and 1 mM ATP, in a final volume of 50 μl. Reactions were incubated for 2 h at 30 °C and stopped by phenol extraction followed by ethanol precipitation. The circularized RNA was then separated in 5 % polyacrylamide gels containing 8 M urea and 1× TBE, and phenol-extracted from gel slices.The retrozyme RNA, with 5′-hydroxyl and 2,3′-phosphodiester termini, was circularized using the chloroplastic isoform of a tRNA ligase from elongena , kindly circRNA, circular RNA; HHR, hammerhead ribozyme; LINE, long interspersed element; LTR, long terminal repeat; MITE, miniature inverted-repeat transposable element; PBS, primer binding site; PPT, polypurine tract; RT, retrotranscriptase; SINE, small interspersed nucleotide element; SMART, small LTR-retrotransposon; TRIM, terminal-repeat retrotransposon in miniature; TSD, target site duplication


N = 989) and (2) prospective (N = 896) students. We developed the Motivations Influencing Course Choice (MICC) questionnaire to measure these motivations. Scales of Helping, Career, Loafing, and Interest showed good psychometric properties, showed validity with respect to general life goals and personality traits, and predicted actual and prospective degree choices. We demonstrated that medical degrees were chosen due to a mixture of Helping and Career, while engineering degrees were associated with Career and low Interest in the degree. The choice of arts and humanities degrees was driven by Interest and low concern about future career, accompanied with high Loafing. We also demonstrated gender differences: females were high in Helping (both samples) and Interest (only in the undergraduate sample) motivation, while males scored higher in Career (only in the undergraduate sample) and Loafing (both samples). The findings can feed into both theoretical accounts of proximal motivation as well as provide help to improve degree programmes at universities and support better career advice.Different people choose undergraduate degrees to study at university for different reasons. To date, there have been limited attempts to identify individual differences in motivation that drive undergraduate degree choice. We identified that people choose university degrees for four reasons: career concerns (Career), intrinsic interest in the subject (Interest), an opportunity to help others (Helping) and because they are looking for an easy option to get into higher education (Loafing). We investigated whether these motivations apply to the choice of undergraduate degree in two samples: (1) undergraduate ( Motivations have been shown to affect real life outcomes: pro-social motivation predicts volunteering, while motivation of achievement predicts career success  and replicated the measure in a sample of prospective university students who attended an Open Day (Sample 2). We used both samples to test the validity of the new developed scales. The study was approved by Ethics Committee of the School of Psychology, University of Nottingham.N = 397), an online from a large campus-based British university  and a pen/paper from the same university as a second sample . Subsamples differed significantly in age and gender, with participants from the subsample P&P being younger than both Online 1 and Online 2 . Subsample P&P had equal gender distribution ; proportion of females was significantly higher in Online 1 and Online 2 . The overall sample was not different in gender  and age  from a typical undergraduate sample. As subsamples differed in age and gender, we controlled for these variables in all subsequent analyses. Some participants did not fill in all of the questionnaires (they were free to withdraw at any point). Participants who did not finish all questionnaires were included in all relevant analyses (where it was possible) as they were not different in characteristics from the rest of participants. 3.3% of the overall sample reported studying medical sciences, 29.1% social sciences, 44.7% sciences, 11.1% arts and humanities and 11.8% engineering.Sample 1 data was collected through an online and pen/paper form of the survey. An opportunity sample of 989 participants from two large UK universities was recruited , incentivised by a prize draw of £75. The overall sample included three opportunity subsamples: an online from a large city-based British university , with an incentive of a prize draw of £75 in Amazon vouchers. 16.2% of the overall sample reported wishing to study medical sciences, 25.8% social sciences, 24.9% sciences, 21.9% arts and humanities and 3.1% engineering.not at all to very much so. Two pilot studies were conducted with opportunity samples of undergraduate students (Ns = 115 and 187), which aimed to ensure that students could relate to the content of the items. Out of initial pool of fifty-five, six items were removed due to a high skew. The full initial list of items is reported in Supplementary Materials. In the undergraduate sample we asked participants about their reasons for undergraduate degree choice using stem “I have chosen this degree because…,” followed by respective reason, e.g., “I was interested in the subject,” while for the open days sample the stem was changed to “I am choosing this degree because…”MICC was developed to measure different undergraduate degree choice motivations. Items were generated corresponding to four themes described in the introduction: helping, career, interest, and loafing. All items were answered on 6-point Likert-type scales with participants judging how relevant each specific reason was for their choice of degree, ranging from N = 494) and the second for Confirmatory Factor Analysis . Comprehensive Exploratory Factor Analysis (CEFA) , the comparative fit index (CFI) and the incremental fit index (IFI). A model with a RMSEA below 0.08 and CFI and IFI greater 0.95 indicates a good fit of the data  to very accurate (9).Reliability of the scales was assessed using Cronbach alphas and mean inter-item correlations. Validity of the MICC scales were assessed through associations with the Aspirations Index  = 0.086 . The remaining four factors all had at least three items with loadings >0.57. These four factors were identified as follows: Helping, Loafing, Interest, and Career  = 0.069 , CFI = 0.95, IFI = 0.95, N = 495] and to Sample 2  = 0.074 , CFI = 0.94, IFI = 0.94, N = 896].The four-factor oblique model, based on the CEFA, demonstrated good fit to Sample 1 , and 0.80 for Sample 2 . With regard to scale homogeneity, the MICs were acceptable  for subscales to measure relatively narrow constructs  are shown in Table t(940) = −5.16, p < 0.0001 Sample 1, d (95% CI) = 0.34 ; t(894) = −4.410, p < 0.0001, d = 0.33  for Sample 2], while males were higher in Career . Females in both samples assigned more importance to Interest in their degree concerns , while choosing what to study compared to male participants. In addition, in both samples males were higher in Loafing Gender differences in motivations to choose undergraduate degrees were consistent with those reported previously in the literature in both samples  with the life goals Fame, Image and Personal Growth, and Emotional stability and Openness, in both samples. There were differences between samples. Confirming predictions, Career correlated positively  with Extraversion, but only in the potential students' sample. In addition, Career correlated positively  with Agreeableness in the undergraduate sample. Career also positively correlated  with life goal Relationships in the undergraduate sample.Using logistic regressions, we investigated how well specific dimensions of the MICC questionnaire predicted the choice of undergraduate degrees. A series of five hierarchical logistic regression tested predictive properties of the MICC scales with respect to undergraduate degree choice with age and gender controlled. The outcome variables for each regression were coded as 1 for the targeted degree type  vs. 0 for all other types. Age and gender were entered first, followed by life goals at Step 2, and the MICC scales at Step 3. We conducted additional analysis where each motivation was added separately at Step 3, and results are presented in Table In line with predictions, the choice of medical degrees was positively associated with Helping and Career motivation in both samples. In both samples Helping motivation explained the highest proportion of variance compared to other motivations. In addition, medicine was negatively associated with Loafing in the potential students' sample. In both samples the choice of science degree was negatively associated with Helping. In addition, in the undergraduate sample, the choice of science degree was positively associated with Interest and Career. There was little difference in the relevant contribution each motivation has made to the explanation of the choice of science degree. The choice of engineering degree was positively associated with Career and negatively with Interest in both samples. In addition, engineering undergraduates reported Helping as low in importance in terms of their degree choice. For the undergraduate sample only, Career was the strongest predictor over other motivations in terms of explained variance. There was no consistency between samples in terms of predictions for social science degrees. Those who were studying social sciences reported Helping as important, and Career and Interest as unimportant reasons for their degree choice, with Career motivation explaining the most of the variance. Those who were planning to study social sciences reported Career and Interest as important when considering the choice of degree, at the same time being low on Loafing. Current and potential students, who chose/were choosing to study arts and humanities degrees, were concerned how interesting the degree was for them (high Interest) and how easy it would be to complete it (high Loafing), while not taking into account career prospects (low Career). In addition, potential students who were planning to study arts and humanities, reported low concern about opportunities to help others (low Helping). In terms of variance explained, for undergraduate students Loafing (high) and Career (low) were the strongest predictors, while for potential students the strongest predictors were Career (low) and Interest (high).Finally, we tested incremental validity of the MICC scales over the life goals with respect to undergraduate degree choice using the results of the regressions described above Table . Age andThe 18-items MICC questionnaire is a new measure of individual differences in undergraduate degree choice motivation. We identified four subscales of the MICC that equate to four domains of motivation: Career, Interest, Loafing, and Helping. The results indicate that the MICC has a clear factor structure and strong psychometric properties. Different subscales of the MICC questionnaire showed predicted associations with life goals and the Big Five domains.Both Helping and Interest correlated positively with all three intrinsic life goals , suggesting that these subscales capture concern for others as well as striving for personal growth, as defined by SDT  confirming that Loafing and Career are aligned with extrinsic motivation. However, they were distinct psychometrically and were associated with different degrees choices. Career, consistently across both samples, positively predicted the choice of medical and engineering degrees, while Loafing predicted the choice of arts and humanities. In addition, Career was positively associated with intrinsic life goals: Personal Growth in both samples and Relationships in the undergraduate sample. Loafing was negatively associated with all three intrinsic life goals: Personal Growth, Relationships, and Community. Furthermore, in terms of personality traits, Loafing was associated negatively with Extraversion, Agreeableness and Conscientiousness, while Career demonstrated an opposite pattern. Thus, those who indicated Loafing as a motivation for their degree choice were less sociable, and were less friendly and more disorganized. Those who indicated Career as a motivation for their degree choice were sociable, friendly, and organized, plus emotionally stable and opened to new experiences.These findings add to the previous theoretical literature on motivation. SDT proposes that intrinsic and extrinsic motivations are the different end of the same continuum, and measuring individual differences on this continuum is sufficient to predict real life outcomes. In our study both intrinsic and extrinsic dimensions were represented by two motivations each: Career and Loafing for extrinsic, and Helping and Interest for intrinsic. Further, all four undergraduate degree choice scales were psychometrically distinct. Based on SDT, we should be able to predict real life outcomes from the extent to which a person is extrinsically or intrinsically motivated. However, it seems that one dimension might be not enough to make a prediction about specific outcome and, in addition, both intrinsic and extrinsic motivations have different expressions. For example, somebody who is intrinsically motivated could have been driven by interest in the subject and chose to study arts, while if they were striving to help others, they might have chosen to study medicine. Furthermore, as our data shows, it is plausible that both an intrinsic and extrinsic type of motivation could drive the choice of the same individual. For example, medical degrees were chosen because of Helping and Career. This is in line with Chemolli and Gagné  who usedOur data supports the multideterministic nature of the choice Pervin, . We demoTheoretically this study contributes a set of “proximal” motivational constructs, which can explain differences in choices within a specific context of career path. Proximal motivations have been discussed in previous research and are more contextualized in relation to choice, compared to generic motivations. Previous researchers discussed the need to measure proximal motivation in addition to more distal constructs, such as life goals and personality traits, in order to improve predictability of the models Elliot, . The curIn addition to differences in personality and life goals, we reported gender differences in degree choice motivation. Male participants reported higher Career (only Sample 1) and Loafing motivation (both samples), while female participants were high in Helping (both samples) and Interest . These findings support previous research in gender specific social roles in terms of Helping and Career, however, it is not clear why there should be gender difference in Interest and Loafing. Further research could investigate whether gender stereotypes predict the choice of undergraduate degree or whether the differences are mediated by other factors, such as the type of chosen degree.The information about what motivates students to choose their degree can be potentially very important for educational institutions and individual departments. For example, it is important to recognize that engineering students currently may not be as concerned about the enjoyment they can gain from studying their future undergraduate degree as lack of interest in the subject leads to the low levels of deep learning . Future studies could benefit by looking into specific subsample of degree types to investigate whether MICC questionnaire holds predictive power for these specific degrees. Finally, we are aware that not all countries' educational systems require students to choose their undergraduate degree prior to the start of their time at university; however it is a common situation in many countries .Overall, the current results demonstrate that individual differences in motivation  are associated with real life choices in the context of the choice of undergraduate degree. The implications of distinct types of contextualized proximal motivation for predicting life changing choices are an exciting research avenue. After establishing the dimensions, it is possible to investigate if those proximal motivations mediate the effects of more distal dispositional motivations (such as approach and avoidance) and personality dispositions on the choice of degree. It will help to establish link between motivational and personality constructs of different levels and actual choices. This will result in better understanding and improved predictions of differences in real life choices.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.