Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

Abstract

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. Picornavirus IRES are organized in structural domains, in which the terminal stem-loops participate in functional RNA-protein interactions. However, the mechanistic role performed by the central domain during internal initiation has not been elucidated yet. Here we show that the foot-and-mouth-disease virus IRES contains a structural motif that serves in vitro as substrate for the Synechocystis sp. RNase P ribozyme, a structure-dependent endonuclease that participates in tRNA precursor processing. Recognition of the IRES substrate was dose dependent, required high magnesium concentration, and resulted in the formation of cleavage products with 59 phosphate and 39 hydroxyl ends. Mapping of the core recognition motif indicated that it overlapped with the apical region of the central domain. Two IRES constructs containing nucleotide substitutions in the apical region of the central domain that reorganized RNA structure displayed an altered pattern of cleavage by the cyanobacterial ribozyme generating new cleavage events in nearby residues. From these data it is inferred that the central domain of the IRES region has evolved a tRNA structural mimicry that renders it a substrate for RNase P ribozyme reaction. Recognition of this motif was affected in defective IRES mutants with a local RNA structure reorganization, suggesting that its structural preservation is required for IRES activity.

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

“…7B). Consistent with the RT-qPCR data shown in Figure 4A, RT stops were located at nucleotides A167, C192, and C211 in the Rz-IRES RNA (nucleotide numbers as in Serrano et al 2007), while a clear RT stop at nucleotide C192 was detected in Rz-GUAG RNA. Interestingly, the primer extension stops were located within the region mapped in vitro as the RNase P recognition motif ).…”

“…cDNA products were analyzed on denaturing 6% acrylamide gels in parallel with a DNA sequence prepared with the same primer. IRES nucleotides (numbered as in Serrano et al 2007) are indicated on the left. Arrows denote the position of RT stops specifically detected in the Rz-IRES and Rz-GUAG RNA.…”

Comparative analysis of the large fragment of the 5′ untranslated region (LF-5′ UTR) of serotype A foot-and-mouth disease virus field isolates from India

“…The 'T 432 CC' motif in the conserved bulge within the 'GNRA' stem-loop, identified as the cleavage site for RNase P, a ribozyme [42] was found as (T/C) CC in this alignment. Neither substitutions in the conserved 'C'-rich loop in domain 3 of FMDV, which is a candidate for PCBP-2 binding in other picornaviruses [43], nor depletion of rabbit reticulocyte lysate of PCBP-2 had significant impact on aphthovirus IRES activity [44,45].…”

Lessons (not) learned from mistakes about translation

“…A frequent problem is deciding the answer first and then constructing a test system to show what one wants to see, as in the selection of a neuronal cell line that showed the expected 9 Some picornavirus IRESs contain sequences that are specifically recognized by RNases (Elgadi and Smiley, 1999;Serrano et al, 2007), but random attack is probably the more common mechanism. Rosenfeld and Racaniello (2005) claimed that cleavage of dicistronic mRNA was ruled out in their study because translation of the 3′ cistron was not reduced by mutations that inactivate the nonsense-mediated decay pathway, but that pathway is not the only source of RNases in yeast.…”