Peter Sarnow

LNA-mediated microRNA silencing in non-human primates

microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg-1 LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes

The exclusive role of the mRNA cap structure in the 40S subunit recruitment process during translation initiation is no longer tenable. The ability of IRES elements and poly(A) tails in yeast to stimulate 40S subunit binding forces a change in this viewpoint. While it is almost certain that the vast majority of mRNAs are translated by 40S subunits scanning from the cap structure, it now seems likely that the 40S subunit can also be brought to the mRNA via an interaction with the mRNA poly(A) tail. Subsequently or simultaneously with this interaction, the cap structure, due to its high affinity for eIF4E, could act as a docking site for the recruited subunit (Figure 3Figure 3). In this model, both the cap structure and the poly(A) tail share the function of 40S subunit recruitment, while the cap structure has the exclusive role of docking the subunit onto a unique position in the mRNA. We note that the role of poly(A) tails in translation in higher eukaryotes is assumed but not yet shown since only the role of poly(A) tails in yeast translation has been thoroughly studied.Cellular IRES elements could replace the role of the cap structure on those mRNAs where the cap is either masked or eIF4E is inactive (Figure 3Figure 3). Recruitment of the 40S subunit to the mRNA by the poly(A) tail could occur prior to or simultaneously with the placement of the subunit at a position on the mRNA determined by the location of the IRES element. In this model, the cap structure and the IRES element have identical functions in the translation process: they assist in recruitment of the 40S subunit to the mRNA, and they provide a loading site for 40S subunits at a unique position on the mRNA.Future work in this exciting area of translation research should help to test the basic tenets of this model. Since many of the central experiments so far have been carried out in cell-free systems and commonly under conditions where mRNA is limiting, it will be important to determine the interplay of the diferent modes of ribosome recruitment under conditions of mRNA competition for initiation factors. Likewise, we do not yet understand much regarding possible differences between the first and subsequent rounds of initiation, and the roles of the cap, IRES and poly(A) tail in ribosome recycling. Along the way, it is anticipated that new insights into how an mRNAs expression can be controlled in the cytoplasm of eukaryotic cells will be uncovered. With more information, it will hopefully become clearer how mRNA sequences, including the 5′ NCR and the 3′ UTR, can regulate an mRNAs expression.

Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells

The adenovirus E1b-58kd tumor antigen has been detected in a physical association with a 54 kilodalton cellular protein in adenovirus-transformed mouse cells. Antibody specific for the E1b-58kd protein coimmunoprecipitates a 54 kd protein from transformed, but not from productively infected, cells. Monoclonal antibody specific for the cellular 54 kd protein coimmunoprecipitates the adenovirus E1b-58kd protein from transformed cell extracts. The same or closely related cellular 54 kd protein, associated with the adenovirus E1b-58kd protein, was present in the SV40 large T antigen-54 kd complex previously detected in SV40-transformed mouse cells. The identity of the 54 kd protein is based on the immunological specificities of the anti-54 kd monoclonal antibodies and partial peptide maps of the 54 kd protein associated with the adenovirus and SV40 tumor antigens. The adenovirus E1b-58kd-54 kd complex, like the SV40 large T antigen-54 kd complex, is heterogeneous in size or mass. While all of the cellular 54 kd protein in the adenovirus-transformed cell extract is found in a complex with the E1b-58kd protein, some of the viral 58 kd antigen is detected in a form not associated with the 54 kd protein. The fact that the adenovirus and Sv40 tumor antigens, both required for transformation, can be found in physical association with the same cellular protein in a transformed cell is a good indication that these two diverse viral proteins share some common mechanisms or functions.

Initiation of Protein Synthesis from the A Site of the Ribosome

Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in the ribosomal P site and a GCU triplet in the A site. P-site mutagenesis revealed that the P site was not decoded, and protein sequence analysis showed that translation initiates at the triplet in the A site. Translational initiation from the A site of the ribosome suggests that the repertoire of translated open reading frames in eukaryotic mRNAs may be greater than anticipated.

Naturally Occurring Dicistronic Cricket Paralysis Virus RNA Is Regulated by Two Internal Ribosome Entry Sites

ABSTRACT Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5′ end of the viral RNA and is more active than the IRES located at the 5′ end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5′ end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Biological basis for restriction of microRNA targets to the 3¢ untranslated region in mammalian mRNAs

MicroRNAs (miRNAs) interact with target sites located in the 3′ untranslated regions (3′ UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3′ UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition

Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non‐shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi‐permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor–cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus‐infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus.

Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes: The IRES Functions as an RNA-Based Translation Factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.

Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus.

The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5′ untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES–40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES–40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.