Identification and characterization of a novel posttranscriptional regulatory element

Posttranscriptional regulation of gene expression is an important mechanism controlling expression of cellular and viral genes. Several key insights into the posttranscriptional control of gene expression were obtained with retroviruses. Here, we focus on the posttranscriptional regulation of gene expression of a mouse complex betaretrovirus - mouse mammary tumor virus (MMTV). MMTV is a retrovirus encoding a Rev-like protein Rem that binds to the Rem responsive element (RmRE) and facilitates nuclear export of intron-containing viral RNA. In contrast to other complex retroviruses, such as HIV-1, the auxiliary protein does not seem to be required for nuclear export of the incompletely spliced env mRNA. Instead, we propose that the MMTV env mRNA carries a cis-acting element (provisionally termed MPPE), localized at the 5`end, which facilitates cytoplasmic accumulation of the transcript. This concept is supported by our recent observations that i) deletion of MPPE (in contrast to RmRE) from subgenomic expression constructs results in a loss of env mRNA in the cytoplasm of transfected cells, and ii) insertion of MPPE into a HIV-1 gag/PR reporter construct counteracts the inhibitory sequences accounting for nuclear retention of the HIV-1 gag/PR mRNA and allows efficient accumulation of the transcript in the cytoplasm. MPPE is predicted to fold (after splicing) at the 5`UTR/env(rem) junction into a long stem loop. The overall geometry of the structure resembles the constitutive transport element (CTE) of MPMV, further supporting the importance of this sequence. In contrast to CTE, however, MPPE does not directly interact with the major nuclear export receptor, Tap. Our attempts to isolate and identify MPPE-interacting factors, resulted in the identification of two cellular proteins containing a conserved cold shock domain, Y-box binding protein (YB-1) and cold shock domain protein A (DBPA). These proteins are multifunctional DNA and RNA-binding proteins shuttling between the nucleus and the cytoplasm and affecting expression of various genes. The aim of the proposed project is to consolidate and expand our preliminary results supporting the model of two distinct RNA elements facilitating cytoplasmic accumulation of MMTV transcripts. During the course of the proposed project we will gain more insight into the mechanism of the MPPE-dependent enhancement of gene expression. We will define functional boundaries and perform a parallel analysis of RNA structure (PARS) to constrain the M-fold based conformation model. Further, we will also investigate the role of the GC-rich element-binding proteins, YB- 1 and DBPA, in the posttranscriptional regulation of expression of MMTV and cellular genes. The localization of MPPE at the 5`end of mRNA resembles the position of a recently identified nuclear export element facilitating cytoplasmic accumulation of mRNAs encoding secretory proteins, for which a cellular RNA export adaptor remains to be identified. Because this factor should recognize an adenine-depleted RNA segment and YB-1 preferentially interacts with GC-rich regions, it seems to be reasonable to hypothesize that the cold shock domain- containing proteins may represent the adaptors interacting with the signal sequence coding region and mediating nuclear export of mRNAs encoding secretory proteins. We believe that the proposed project will unravel some new aspects of posttranscriptional regulation of gene expression and the gained knowledge may also be valuable for the future translational approaches.

Retroviruses served as model organisms and contributed to our understanding of how cellular RNA molecules traffic from the nucleus to the cytoplasm. In fact, the two major RNA export pathways (Tap/NXF1; CRM1) have been discovered using retroviruses such as HIV-1 and MPMV. Like retroviral RNAs, cellular mRNAs have to exit the nucleus, travel to their final destination where they are translated to proteins. A vast majority of cellular primary transcripts are subjected to alternative splicing resulting in the generation of pools of spliced RNA variants (many of which contain introns) before they are exported from the nucleus. How the intron-containing spliced variants overcome the cellular surveillance system, which prevents the intron-containing RNAs from being exported, remains unknown. Here, we report that a retrovirus mouse mammary tumor virus (MMTV) makes use of splicing to form a potent RNA element that drives the export of a singly-spliced, intron-containing RNA from the nucleus. More specifically, juxtaposition of two exons in the spliced RNA results in the generation of an element reminiscent of the constitutive transport element (CTE) of MPMV. The RNA element, named MPPE, is highly structured as determined by using a mfold prediction algorithm coupled with nuclease probing followed by a deep sequencing. Its presence at the 5end of the singly-spliced env mRNA overrules the CRM1 export signal present at the 3 end and directs the nucleo-cytoplasmic translocation via a CRM1-independent pathway. A newly developed GFP-based nuclear export reporter assay revealed that MPPE represents a potent RNA export element that outperforms the CTE or RRE/Rev exports signals. Furthermore, the reporter assay may be used for the identification of similar RNA export elements in a genome wide scale. Indeed, cloning of a library of 400 nt long RNA fragments derived from cellular mRNAs showed an upregulation of the overall GFP expression levels similar to those found for MPPE. Identification of the RNA elements facilitating mRNA export and in turn the GFP expression is under way. In summary we show for the first time that i) a single mRNA may contain two distinct RNA export elements; ii) juxtaposition of two exons forms an RNA export function; iii) splicing may form more potent export elements that those known so far; iv) a novel GFP-based reporter assay may be used in genome-wide studies aiming to the identify cellular cis-acting RNA export functions. The work presented here paves the way for the understanding of how alternative splicing is regulated.