Fulvia Bono

Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription–polymerase chain reaction (RT‐PCR) in 341 consecutive anti‐HCV–positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations ≤5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome. (HEPATOLOGY 1995;21:285–290.)

Assembly, disassembly and recycling: the dynamics of exon junction complexes.

Efficient gene expression requires that, during their lifetime, mRNAs associate with different sets of RNA binding proteins to form messenger ribonucleoprotein particles (mRNPs). The protein components of mRNPs are essential for the correct post-transcriptional function and regulation of mRNAs. mRNPs are constitutively remodeled during the maturation of the mRNA in the nucleus and downstream steps in the cytoplasm, and can also change depending on the cellular environment. Here we review the current understanding of the biochemical and structural aspects of a central mRNP component and regulator, the exon junction complex (EJC).

Molecular insights into the interaction of PYM with the Mago–Y14 core of the exon junction complex

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago–Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago–Y14. Here we show that PYM is a cytoplasmic RNA‐binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago–Y14 by means of its N‐terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense‐mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD.

Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex.

In mammals, Up-frameshift proteins (UPFs) form a surveillance complex that interacts with the exon junction complex (EJC) to elicit nonsense-mediated mRNA decay (NMD). UPF3b is the component of the surveillance complex that bridges the interaction with the EJC. Here, we report the 3.4 Å resolution crystal structure of a minimal UPF3b-EJC assembly, consisting of the interacting domains of five proteins (UPF3b, MAGO, Y14, eIF4AIII, and Barentsz) together with RNA and adenylyl-imidodiphosphate. Human UPF3b binds with the C-terminal domain stretched over a composite surface formed by eIF4AIII, MAGO, and Y14. Residues that affect NMD when mutated are found at the core interacting surfaces, whereas differences between UPF3b and UPF3a map at peripheral interacting residues. Comparison with the binding mode of the protein PYM underscores how a common molecular surface of MAGO and Y14 recognizes different proteins acting at different times in the same pathway. The binding mode to eIF4AIII identifies a surface hot spot that is used by different DEAD-box proteins to recruit their regulators.

Molecular epidemiology of hepatitis C virus infection among intravenous drug users

BACKGROUND/AIMS The clinico-pathological features of hepatitis C virus infection in intravenous drug users are different from those found in other hepatitis C virus-infected patients. Our aim was to test whether specific viral variants circulate within this particular patient population. METHODS We studied the distribution of hepatitis C virus genotypes in 90 drug addicts and 484 controls, according to the method described by Okamoto. RESULTS Hepatitis C virus type 1a and 3a infections were more frequent among intravenous drug users than in 125 age-matched controls (48.8% and 21.1% vs 17.6% and 11.2%), accounting for the majority of infections in intravenous drug users. Analysis of hepatitis C virus genotypes according to age showed that, in the general population, hepatitis C virus types 1a and 3a were more prevalent among patients younger than 40 years of age than in older individuals (17.6% and 11.2% vs 1.4% and 0.6%). CONCLUSIONS These findings suggest that hepatitis C virus types 1a and 3a were recently introduced in Italy, presumably via needle-sharing among intravenous drug users, and from this reservoir they are extending to the general population, particularly among younger subjects.

Nuclear Import Mechanism of the EJC Component Mago-Y14 Revealed by Structural Studies of Importin 13

Mago and Y14 are core components of the exon junction complex (EJC), an assembly central to nonsense-mediated mRNA decay in humans and mRNA localization in flies. The Mago-Y14 heterodimer shuttles between the nucleus, where it is loaded onto specific mRNAs, and the cytoplasm, where it functions in translational regulation. The heterodimer is imported back into the nucleus by Importin 13 (Imp13), a member of the karyopherin-beta family of transport factors. We have elucidated the structural basis of the Mago-Y14 nuclear import cycle. The 3.35 A structure of the Drosophila Imp13-Mago-Y14 complex shows that Imp13 forms a ring-like molecule, reminiscent of Crm1, and encircles the Mago-Y14 cargo with a conserved interaction surface. The 2.8 A structure of human Imp13 bound to RanGTP reveals how Mago-Y14 is released in the nucleus by a steric hindrance mechanism. Comparison of the two structures suggests how this unusual karyopherin might function in bidirectional nucleocytoplasmic transport.

Crystal structure of a minimal eIF4E–Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.

Structure of Importin13–Ubc9 complex: nuclear import and release of a key regulator of sumoylation

Importin13 (Imp13) is an unusual β‐karyopherin that is able to both import and export cargoes in and out of the nucleus. In the cytoplasm, Imp13 associates with different cargoes such as Mago‐Y14 and Ubc9, and facilitates their import into the nucleus where RanGTP binding promotes the release of the cargo. In this study, we present the 2.8 Å resolution crystal structure of Imp13 in complex with the SUMO E2‐conjugating enzyme, Ubc9. The structure shows an uncommon mode of cargo–karyopherin recognition with Ubc9 binding at the N‐terminal portion of Imp13, occupying the entire RanGTP‐binding site. Comparison of the Imp13–Ubc9 complex with Imp13–Mago‐Y14 shows the remarkable plasticity of Imp13, whose conformation changes from a closed ring to an open superhelix when bound to the two different cargoes. The structure also shows that the binding mode is compatible with the sumoylated states of Ubc9. Indeed, we find that Imp13 is able to bind sumoylated Ubc9 in vitro and suppresses autosumoylation activity in the complex.

Structural basis for the nuclear export activity of Importin13

Importin13 (Imp13) is a bidirectional karyopherin that can mediate both import and export of cargoes. Imp13 recognizes several import cargoes, which include the exon junction complex components Mago‐Y14 and the E2 SUMO‐conjugating enzyme Ubc9, and one known export cargo, the translation initiation factor 1A (eIF1A). To understand how Imp13 can perform double duty, we determined the 3.6‐Å crystal structure of Imp13 in complex with RanGTP and with eIF1A. eIF1A binds at the inner surface of the Imp13 C‐terminal arch adjacent and concomitantly to RanGTP illustrating how eIF1A can be exported by Imp13. Moreover, the 3.0‐Å structure of Imp13 in its unbound state reveals the existence of an open conformation in the cytoplasm that explains export cargo release and completes the export branch of the Imp13 pathway. Finally, we demonstrate that Imp13 is able to bind and export eIF1A in vivo and that its function is essential.

Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability

BackgroundAlvinella pompejana is an annelid worm that inhabits deep-sea hydrothermal vent sites in the Pacific Ocean. Living at a depth of approximately 2500 meters, these worms experience extreme environmental conditions, including high temperature and pressure as well as high levels of sulfide and heavy metals. A. pompejana is one of the most thermotolerant metazoans, making this animal a subject of great interest for studies of eukaryotic thermoadaptation.ResultsIn order to complement existing EST resources we performed deep sequencing of the A. pompejana transcriptome. We identified several thousand novel protein-coding transcripts, nearly doubling the sequence data for this annelid. We then performed an extensive survey of previously established prokaryotic thermoadaptation measures to search for global signals of thermoadaptation in A. pompejana in comparison with mesophilic eukaryotes. In an orthologous set of 457 proteins, we found that the best indicator of thermoadaptation was the difference in frequency of charged versus polar residues (CvP-bias), which was highest in A. pompejana. CvP-bias robustly distinguished prokaryotic thermophiles from prokaryotic mesophiles, as well as the thermophilic fungus Chaetomium thermophilum from mesophilic eukaryotes. Experimental values for thermophilic proteins supported higher CvP-bias as a measure of thermal stability when compared to their mesophilic orthologs. Proteome-wide mean CvP-bias also correlated with the body temperatures of homeothermic birds and mammals.ConclusionsOur work extends the transcriptome resources for A. pompejana and identifies the CvP-bias as a robust and widely applicable measure of eukaryotic thermoadaptation.ReviewerThis article was reviewed by Sándor Pongor, L. Aravind and Anthony M. Poole.