Andre Furger

Integrating mRNA Processing with Transcription

The messenger RNA processing reactions of capping, splicing, and polyadenylation occur cotranscriptionally. They not only influence one anothers efficiency and specificity, but are also coordinated by transcription. The phosphorylated CTD of RNA polymerase II provides key molecular contacts with these mRNA processing reactions throughout transcriptional elongation and termination.

U1 snRNA associates with TFIIH and regulates transcriptional initiation

Diverse classes of noncoding RNA, including small nuclear RNAs (snRNAs), play fundamental regulatory roles at many stages of gene expression. For example, recent studies have implicated 7SK RNA and components of the splicing apparatus in the regulation of transcriptional elongation. Here we present the first evidence of the involvement of an snRNA in the regulation of transcriptional initiation. We demonstrate that TFIIH, a general transcription initiation factor, specifically associates with U1 snRNA, a core-splicing component. Analysis of the TFIIH-dependent stages of transcription in a reconstituted system demonstrates that U1 stimulates the rate of formation of the first phosphodiester bond by RNA polymerase II. In addition, a promoter-proximal 5′ splice site recognized by U1 snRNA stimulates TFIIH-dependent reinitiation of productive transcription. Our results suggest that U1 snRNA functions in regulating transcription by RNA Polymerase II in addition to its role in RNA processing.

Polypyrimidine Tract Binding Protein Modulates Efficiency of Polyadenylation

ABSTRACT Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3′ end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signals pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3′ end processing.

Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3′-end-seq

Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3′-end regions of transcripts (3′-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3′-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3′-end formation in the intestine.

The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation

ABSTRACT Maximal gene expression in retroviruses requires that polyadenylation in the 5′ long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5′ LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5′ LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5′ LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

Analysis of U1 Small Nuclear RNA Interaction with Cyclin H

TFIIH is a general transcription and repair factor implicated in RNA polymerase II transcription, nucleotide excision repair, and transcription-coupled repair. Genetic defects in TFIIH lead to three distinct inheritable diseases: xeroderma pigmentosa, Cockayne syndrome, and trichothiodystrophy, with xeroderma pigmentosa patients being highly susceptible to skin cancer. Earlier data revealed that the cyclin H subunit of TFIIH associates with U1 small nuclear RNA, a core-splicing component. In addition to its role in RNA processing U1 small nuclear RNA also regulates diverse stages of transcription by RNA polymerase II both in vivo and in vitro, including abortive initiation and re-initiation. Here we identify structural components of U1 and cyclin H implicated in the direct interaction and show how they affect function. Because of unique features of cyclin H we have developed a new methodology for mapping RNA interaction with the full-length cyclin H polypeptide based on electrospray ionization tandem mass spectrometry. We also demonstrate the importance of U1 stem-loops 1 and 2 for the interaction with cyclin H. Functional assays implicate the identified interaction with U1 in regulation of the activity of the cyclin H associated kinase CDK7.

Two G-Rich Regulatory Elements Located Adjacent to and 440 Nucleotides Downstream of the Core Poly(A) Site of the Intronless Melanocortin Receptor 1 Gene Are Critical for Efficient 3′ End Processing

ABSTRACT Cleavage and polyadenylation is an essential processing reaction required for the maturation of pre-mRNAs into stable, export- and translation-competent mature mRNA molecules. This reaction requires the assembly of a multimeric protein complex onto a bipartite core sequence element consisting of an AAUAAA hexamer and a GU/U-rich downstream sequence element. In this study we have analyzed 3′ end processing of the human melanocortin 1 receptor gene (MC1R). The MC1R gene is an intron-free transcription unit, and its poly(A) site lacks a defined U/GU-rich element. We describe two G-rich sequence elements that are critical for efficient cleavage at the MC1R poly(A) site. The first element is located 30 nucleotides downstream of the cleavage site and acts as an essential closely positioned enhancer. The second G-rich region is positioned more than 440 nucleotides downstream of the MC1R processing site and is instrumental for optimal processing efficiency. Both G-rich sequences contain clusters of heterogeneous nuclear ribonucleoprotein binding motifs and act together to enhance cleavage at the MC1R poly(A) site.

α-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response.

Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.

Regulation of transcription termination in the nematode Caenorhabditis elegans

The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3′ flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination.