Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes

Abstract

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.

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

“…Thus, within the 7.3-Å cryo-EM reconstruction of the CrPV IGR IRES-80S ribosome complex, domain 3 occupies the 40S subunit A site (Fig. 4A), also in agreement with earlier low-resolution cryo-EM observations (43). This observation suggests that initiation begins with domain 3 bound to the small subunit A site, followed by translocation to the P site, followed by the steps discussed above.…”

“…stem (Fig. 3A), orienting the distal end of domain 3 toward the E site, where domains 1 and 2 are observed in cryo-EM reconstructions of complexes containing complete IGR IRES RNAs bound to 80S ribosomes (23,43). This orientation, similar to that of a P/E hybrid state tRNA (46), would potentially allow an A site tRNA to enter the A/P hybrid state without steric clash (Fig.…”

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

“…Although uncommon, there are examples of transcripts with specific requirement for large subunit proteins during translation initiation. For example, during initiation on the CrPV IRES, the RNA must associate with rpL1 for subunit joining of the 60S to the 40S and correct ribosome positioning (43,44). Furthermore, by studying mice with a short tail phenotype, rpL38 was found to be required for 80S formation on Homeobox mRNAs and thus correct tissue patterning during development (45).…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…On the other hand, structural studies performed on the HCV IRES and the IGR of members of the family Dicistroviridae have shown the capacity of these IRES elements to be accommodated in the interface of the ribosomal subunits (Spahn et al, 2001;Boehringer et al, 2005;Schuler et al, 2006;Pfingsten et al, 2006). Although the IGR and the HCV IRES elements exhibit different structural organization (Kieft et al, 2002;Rijnbrand et al, 2004;Jan & Sarnow, 2002;Nishiyama et al, 2003) and their binding sites in the ribosomal subunit are different, similar conformational changes are induced in the 40S ribosomal subunit (Spahn et al, 2004). This finding opens the possibility that IRES elements could share the property of having a universal structural IRES motif (USIM) that could mediate its direct interaction with the 40S subunit.…”

“…In marked difference to any other IRES described to date, the IGRs of dicistroviruses assemble initiation complexes in the absence of any eIFs (Table 1) (Spahn et al, 2004;Wilson et al, 2000a). This RNA sequence establishes direct contacts with ribosomal proteins (Schuler et al, 2006).…”