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Contents
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We Keep Learning from Retroviruses
Research over approximately the past 20 years has revealed that many retroviruses depend on elaborate mechanisms for nucleocytoplasmic export of their unspliced, full-length RNA. These transcripts encode the Gag-pol polyprotein and, in addition, serve as genomic RNA to be packaged into progeny virions in the cytoplasm. Studies of the molecular biology of HIV-1 have been instrumental for major discoveries in the field of mRNA metabolism and macromolecule transport. In HIV-1 and other lentiviruses, this process depends on the Rev protein, which is essential for the production of structural proteins and infectious virions (Figure 1). Rev promotes export and expression of gag/pol and env mRNAs by binding to the cis-acting RNA recognition signal, the Rev-responsive element (RRE). A similar mechanism is essential for human T-cell leukemia virus (HTLV) and human endogenous retrovirus (HERV-K) expression. Rev and its functional homologs, as well as many cellular proteins, share a leucine-rich nuclear export signal, which is recognized by the cellular export receptor CRM1 thereby linking the mRNP (mRNA-protein) cargo to the nuclear pore complex. The discovery of the Rev export pathway paved the way to understanding the trafficking of cellular proteins such as MDM2, MAPKK, PKI, and others. Figure 1. Distinct export pathways from the nucleus. CRM1 and NXF1 represent two key export pathways from the nucleus. Studies of retroviruses and retroelements have been critical for their discovery. CRM1 is essential for the Rev-mediated HIV mRNA export and expression. NXF1 is the molecular link between the constitutive transport element (CTE)– and RNA transport element (RTE)–containing mRNAs and the nuclear pore complex. The key factors mediating mRNA export of retrovirus and retroelement mRNA export also promote transport of cellular mRNAs and proteins. Rev, HIV-1 protein essential for the production of structural proteins and infectious virions; RRE, Rev-responsive element; SRV, simian type D retrovirus. Expression of simian type D retrovirus (SRV/MPMV) depends on the cellular trans-acting factor TAP/NXF1, which binds to the cis-acting constitutive transport element (CTE) (Figure 1). The extended stem-loop structure of CTE is conserved among all type D species. A CTE-related element was found in a subgroup of rodent intracisternal A particle retroelements (IAP), and more than 100 CTE-related elements are present in the mouse genome. The cellular NXF1 is not only the export receptor of CTE-containing RNA, but most importantly, it is the key export factor for cellular mRNAs, a function that is conserved among eukaryotes. We discovered another potent RNA transport element (RTE, Figure 1), linked to a “fossilized” mouse IAP (Nappi F et al. J Virol 75: 4558–69, 2001). RTE is functionally similar, but structurally unrelated, to CTE and also functions in many cell types of different species, indicating that its export factor(s) are widely expressed and evolutionarily conserved. RTE does not bind the export factor NXF1 and mediates mRNA export via interactions with other still unknown cellular factor(s). Similar to CTE, RTE depends on a conserved cellular transport mechanism, which makes this mRNA export element a valuable tool for further understanding the mRNA nucleocytoplasmic transport. Here, using computer prediction supported by experimental RNA structure analysis, we found that RTE folds into a novel, extended RNA secondary structure (Figure 2) (Smulevitch S et al. J Virol 79: 2356–65, 2005). Detailed mutational analysis revealed that the minimal RTE contains four internal stem-loops that are indispensable for function in mammalian cells. Therefore, the RTE depends on a complex secondary structure, which is important for the interaction with cellular export factor(s). Sequence similarity analyses revealed that in addition to more than 100 identical RTEs, there are more than 3,000 RTE-related elements in the mouse genome, which share at least 70% sequence identity and which can be found on all the chromosomes. The predicted key structural features of RTE are preserved among these related elements, consistent with their functional importance. Based on their sequence and structure, these elements form four subgroups (Figure 2, structures A through D). Figure 2. Family of RTE-related elements in the mouse genome. The 226-nucleotide (nt)–spanning RTE was used to identify related elements in the mouse genome. Based on structure and sequence, these elements form four groups. The top panel shows the alignment of the RTE and a representative member of each group of RTE-related elements found in GenBank entries al663101 (Group B), al671215 (Group C), and ac003061 (Group D). The shaded areas indicate the loops defined for RTE. Phylogenetic tree and comparison of the identified RTE structure and the predicted secondary structures for the RTE-related elements are shown (bottom panel). SL I, II, III, and IV indicate the identified stem-loop (SL) structures with the RTE. Our research on posttranscriptional elements has further given new insights into the biology of retroelements and their potential effect to alter cellular gene expression. We found that IAP retroelements are more complex than previously thought and that they fall into at least two subfamilies depending on the presence of either the CTE- or the RTE-related RNA export elements. Using an active IAP, we recently found that removal of its RTE leads to abolishment of retrotransposition. This experiment showed for the first time that posttranscriptional control is essential not only for retroviruses but also for long terminal repeat (LTR) retroelements. Our findings suggest that active RNA export elements are inserted into genes via retrotransposition, and can thereby affect the posttranscriptional regulation of cellular gene expression. The presence of the many RTEs in the genome provides us with important new information about posttranscriptional regulation, genome organization, genome evolution, and the potential of IAPs to affect cellular gene expression, which may lead to carcinogenesis.
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etroviruses
gave us oncogenes, and their study helped in the elucidation of molecular mechanisms
of carcinogenesis. However, this is not all. Retroviruses and retroelements
keep us busy in the discovery and the refining of our understanding of basic
mechanisms mediating gene expression. Posttranscriptional regulation is a critical
step in mRNA metabolism that controls the levels of gene expression of both
viral and cellular genes. The detailed analysis of the fundamental cellular
processes that guide the complex assembly of mRNA and proteins and their transport
from the nucleus to the cytoplasm is essential for understanding the regulation
of gene expression. 
