Bernd-Joachim Benecke

In vitro translation of drosophila heat-shock and non-heat-shock mRNAs in heterologous and homologous cell-free systems

Upon heat shock, Drosophila Kc cells still contain normal cellular messenger RNAs in the cytoplasm. The distribution of these 25 degrees C mRNAs between polysomes and the postpolysomal fraction of heat-shocked cells appears unaltered as compared with control cells. The translatability of these normal cellular messages isolated from heat-shocked and non--heat-shocked Kc cells is unaltered when analyzed by in vitro translation in the rabbit reticulocyte lysate. In contrast, homologous cell-free translation systems obtained from Kc cells effectively discriminate between the in vitro translation of normal cellular messages and heat-shock--specific mRNAs. In particular, a cell-free system from heat-shocked Drosophila Kc cells almost completely shuts down the translation of 25 degrees C messenger RNA species, whereas the translatability of heat-shock--specific messenger RNA appears to be unaffected.

Invertebrate 7SK snRNAs.

Abstract7SK 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.

The vertebrate 7S K RNA separates hagfish (Myxine glutinosa) and lamprey (Lampetra fluviatilis).

Abstract. 7S RNA sequences from the hagfish (Myxiniformes) and lamprey (Petromyzontiformes) were cloned and analyzed. In both species, 7S L RNA (also designated SRP RNA, since it represents the RNA constituent of the signal recognition particle) was clearly detectable. The sequence similarity between the two species was 86%, compared with about 75% similarity between either of these species and mammals. 7S K RNA was also cloned from the lamprey. The similarity between the 7S K RNA of the lamprey and that of mammals was 68%. Interestingly, several interspersed elements were found with nearly 100% similarity compared with mammals. In contrast to the lamprey, no 7S K RNA-related sequences were detectable among hagfish RNA, neither in northern blots nor with the PCR assay. In view of the significant conservation between the 7S K RNA of lamprey and that of mammals (human), this unexpected result clearly separates lamprey and hagfish. In addition, the lack of detectable 7S K RNA sequences in an outgroup, such as amphioxus, indicates that these results do not reflect an autapomorphy of hagfish. Therefore, our data provide additional support to the notion of a sister group relationship between Petromyzontiformes and gnathostomous vertebrates to the exclusion of Myxiniformes.

RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter

Abstract Small interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more active in vivo than other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.

7SK snRNA-mediated, gene-specific cooperativity of HMGA1 and P-TEFb

7SK small-nuclear RNA has been shown to negatively regulate P-TEFb transcription elongation on the one hand and control HMGA1 transcription initiation and chromatin remodeling on the other. The non-coding 7SK RNA thereby directly interacts with both factors through different regions. While the loop 2 of the RNA specifically binds to the first HMGA1 A/T hook, thereby competing with DNA binding to the same domain, loops 1, 3 and 4 are involved in P-TEFb interaction. This raises the question of whether HMGA1 and P-TEFb cooperate during gene transcription. Using transcriptome profiling, we have identifed genes that are oppositely regulated by 7SK RNA over-expression versus shRNA mediated knock-down. Inhibition of P-TEFb by competitive expression of a dominant-negative Cdk9 protein leads to highly similar changes in global gene expression as the over-expression of 7SK RNA, confirming the importance of P-TEFb inhibition by 7SK RNA. Furthermore, we have similarly assembled genes affected concomitantly by HMGA1 over-expression. HMGA1 and P-TEFb, in the case of select target genes, show strong cooperation in transcriptional activation. Finally, we provide evidence for 7SK RNA complexes containing simultaneously HMGA1 and P-TEFb. 7SK RNA thus establishes gene-dependent plasticity between HMGA1 chromatin remodeling and transcription initiation and P-TEFb transcription elongation.

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

HMGA1-dependent and independent 7SK RNA gene regulatory activity

The small nuclear 7SK RNA negatively controls transcription by inactivating positive transcription elongation factor b (P-TEFb) and is an integral component of Tat-dependent and independent HIV-1 transcription initiation complexes. 7SK RNA has recently been shown to also directly control HMGA1 transcription activity. HMGA1 is a master regulator of gene expression and its deregulation is associated with virtually any type of human cancer. The degree of HMGA1 over-expression thereby correlates with tumor malignancy and metastatic potential. 7SK snRNA directly interacts through its loop2 (7SK L2) with the first A/T-hook DNA binding motif of HMGA1. We have developed several 7SK L2 RNA chimera with the Epstein Barr Virus expressed RNA 2 (EBER2) to target HMGA1 function in transcription regulation. The efficiency of interfering with HMGA1 transcription activity by the chimeric 7SK L2 — EBER2 fusions by large exceeds the efficiency of 7SK wild-type RNA due to the stronger EBER2 promoter activity. Furthermore, the 7SK L2 — EBER2 chimera do not interfere with P-TEFb controlled transcription elongation or the formation of 7SK sn/hnRNPs. The comparison of the effects of wild-type 7SK RNA on cellular transcriptome dynamics with those induced by the two 7SK L2 mutants as well as the changes in gene expression following inhibition of HMGA1 allow the identification and characterization of HMGA1- dependent and independent effects of 7SK snRNA. We furthermore also present evidence for P-TEFb and HMGA1-independent 7SK RNA L2 regulatory activity.

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

BackgroundLittle is known about the physiological role of the EBER1 and 2 nuclear RNAs during Epstein Barr viral infection. The EBERs are transcribed by cellular RNA Polymerase III and their strong expression results in 106 to 107 copies per EBV infected cell, making them reliable diagnostic markers for the presence of EBV. Although the functions of most of the proteins targeted by EBER RNAs have been studied, the role of EBERs themselves still remains elusive.FindingsThe cellular transcription response to EBER2 expression using the wild-type and an internal deletion mutant was determined. Significant changes in gene expression patterns were observed. A functional meta-analysis of the regulated genes points to inhibition of stress and immune responses, as well as activation of cellular growth and cytoskeletal reorganization as potential targets for EBER2 RNA. Different functions can be assigned to different parts of the RNA.ConclusionThese results provide new avenues to the understanding of EBER2 and EBV biology, and set the grounds for a more in depth functional analysis of EBER2 using transcriptome activity measurements.

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

The 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/HEXIM1-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.

Expression levels of heat shock factors are not functionally coupled to the rate of expression of heat shock genes

The expression patterns of two mammalian heat shock factors (HSFs) were analysed in cell systems known to reflect an altered heat shock response. For being able to discriminate between the two closely related factors HSF 1 and HSF 2, specific cDNA sequences were cloned and used to generate antisense RNAs as hybridization probes. In general, in various cell lines expression of the two heat shock factors was clearly different. These expression patterns of the HSF genes were not influenced by retinoic acid-induced differentiation of human NT2 and mouse F9 teratocarcinoma cells. Generally, HSF 2 expression was extremely low, whereas the significantly higher expression of HSF 1 revealed cell specific differences. The highest expression rates of both HSFs were observed in 293 cells. To examine whether these high levels are involved in the constitutive expression of heat shock genes in these cells, we analysed the binding pattern of 293 cell proteins to the heat shock elements (HSEs). As with other cells, HSE-binding activity in 293 cells was only observed after heat shock treatment. This points to an HSE-independent way for high level expression of heat shock genes in these cells.