The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function

Abstract

Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.Post-transcriptional control is essential for expression of cellular and viral mRNAs, involving complex interactions of messenger ribonucleoproteins with transport receptors and components of the nuclear pore complex. Retroviruses depend on specialized transport mechanisms for nuclear export of full-length mRNA in their unspliced form because this transcript encodes the Gag-Pol polyprotein and additionally serves as the genomic RNA packaged into progeny virions in the cytoplasm. For HIV and all lentiviruses, the human T-cell leukemia virus family, endogenous human retroviruses, and mouse mammary tumor virus, transport of full-length mRNA depends on specific cis-acting RNA export signals that bind viral trans-acting factors and link the mRNA with the cellular CRM1 (chromosome region maintenance 1) export receptor (1, 2). In contrast, export and expression of the primary transcript of simple retroviruses and retroelements depend solely on cellular export machinery. Simian retrovirus and MasonPfizer monkey virus contain the cis-acting constitutive transport element (CTE) 3 (3, 4), and some rodent intracisternal A-particle LTR retroelements contain a CTE-like element designated the RNA transport element (RTE) (5, 6). These export elements represent interaction sites for cellular factors that provide a molecular link to the NXF1 (nuclear export factor 1) export receptor (7-9). Inactivating the retroviral export machinery by removing the cis-acting RNA element or trans-acting protein factors results in nuclear retention of the primary transcript and consequently impaired virus or retroelement production.Like the intracisternal A-particle LTR, the type D murine LTR retrotransposon (musD) and its derivative, ETn (early transposon), are mobile elements that share ancestry and genomic organizatio...

Automated 3D structure composition for large RNAs

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Figure 5.Prediction of the tertiary structure of the MusD RNA transport element. ( A ) The reported secondary structure (43) in the dot-bracket notation. First-order interactions are represented by square brackets, while the second-order interactions are represented by curl brackets (highlighted in green).

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Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…Similar C-and U-rich loop regions in other RNAs have been found to have low reactivity toward SHAPE reagents (Legiewicz et al 2010), in part because of the slightly lower reactivity of C and U nucleotides compared with A and G (Wilkinson et al 2009). Our Tb 3+ data, however, exhibit the same protection of these nucleotides as 1M7.…”

Correlating SHAPE signatures with three-dimensional RNA structures

“…Chemical probing techniques such as hydroxyl radical footprinting (Tullius and Dombroski 1985;Tullius and Greenbaum 2005) or in-line probing (Regulski and Breaker 2008;Wakeman and Winkler 2009a,b) are important approaches for RNA structure determination. In recent years, the development of innovative methods such as SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) (Merino et al 2005;Wilkinson et al 2008), SHAMS (selective 29-hydroxyl acylation analyzed by mass spectrometry) (Turner et al 2009), and antisense interfered aiSHAPE (Legiewicz et al 2010) for probing secondary and tertiary structure of RNA molecules has opened new avenues for RNA research. Certain hydroxylselective electrophiles, such as N-methylisatoic anhydride (NMIA) (Merino et al 2005) and 1-methyl-7-nitroisatoic anhydride (1M7) (Mortimer and Weeks 2007), readily react with 29-OH groups of ribose moieties at nucleotide positions that are single-stranded and unconstrained.…”