EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection

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Invertebrate 7SK snRNAs

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3′ and 5′ stem-loop motifs, and provide evidence for a third, structurally well-conserved domain

Efficient transcription of the EBER2 gene depends on the structural integrity of the RNA

A 3′-truncated EBER2 RNA gene, although containing all previously identified promoter elements, revealed drastically reduced transcription rates in vitro and in vivo when fused to a heterologous terminator sequence. Inactivations were also observed with double point mutations affecting 5′- or 3′-end sequences of the EBER2 gene. However, wild-type activity of these mutants could be restored by compensatory mutations of the opposite strand of the EBER2 RNA sequence. A similar rescue was achieved with the 3′-truncated EBER2 gene, if the heterologous terminator was adapted for complementarity to the initiator element of the construct. Yet, double-strandedness alone of the RNA ends was not sufficient for high transcriptional activity of these gene constructs. Rather, the use of a nonrefoldable spacer, separating the 5′- and 3′-stem–loop structures, demonstrated that spatial proximity of the ends of EBER2 RNA was required. Furthermore, decay kinetics of wild-type and mutant RNA synthesized in vitro indicated that the effects observed could not be explained by altered transcript stability. Finally, single-round transcription confirmed that the reduced expression of mutant genes was not caused by decreased primary initiation reactions. In addition, differential sarcosyl concentrations demonstrated that the rate of reinitiation clearly was affected with the mutant EBER2 genes. Together, these results indicate that the secondary structure of this viral RNA represents a major determinant for efficient transcription of the EBER2 gene by host cell RNA polymerase III

HMGA1 directly interacts with TAR to modulate basal and Tat-dependent HIV transcription

International audienceThe transactivating response element (TAR) of human immunodeficiency virus 1 (HIV-1) is essential for promoter transactivation by the viral transactivator of transcription (Tat). The Tat-TAR interaction thereby recruits active positive transcription elongation factor b (P-TEFb) from its inactive, 7SK/HE XIM1-bound form, leading to efficient viral transcription. Here, we show that the 7SK RNA-associating chromatin regulator HMGA1 can specifically bind to the HIV-1 TAR element and that 7SK RNA can thereby compete with TAR. The HMGA1-binding interface of TAR is located within the binding site for Tat and other cellular activators, and we further provide evidence for competition between HMGA1 and Tat for TAR-binding. HMGA1 negatively influences the expression of a HIV-1 promoter-driven reporter in a TAR-dependent manner, both in the presence and in the absence of Tat. The overexpression of the HMGA1-binding substructure of 7SK RNA results in a TAR-dependent gain of HIV-1 promoter activity similar to the effect of the shRNA-mediated knockdown of HMGA1. Our results support a model in which the HMGA1/TAR interaction prevents the binding of transcription-activating cellular co-factors and Tat, subsequently leading to reduced HIV-1 transcription

Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli