Dirk Eick

Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription.

The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique domain composed of heptapeptide repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the CTD code. In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response

High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments.

Heptad-specific phosphorylation of RNA polymerase II CTD.

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.

Cyclin-dependent kinase 9 links RNA Polymerase II transcription to processing of ribosomal RNA

Background: Processing of ribosomal RNA is sensitive to Cdk inhibitors. Results: Inhibition of Cdk9 activity blocks 47 S rRNA processing and stabilizes a 3′ extended 47 S primary transcript. Defective 3′ processing negatively feeds back on RNAPI transcription. Conclusion: Cdk9 facilitates processing of 47 S rRNA by RNAPII-dependent synthesis of U8 snoRNA. Significance: Cdk9 may be a critical regulator of rRNA processing to harmonize RNAPII transcription activity with a ribosome biogenesis rate. Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

Getting Access to Low-Complexity Domain Modifications.

Low-complexity (LC) domains regulate the aggregation and phase transition of proteins in a modification-dependent manner. The study of LC domain modifications has now become feasible, as shown by genetic variants of the carboxy-terminal domain (CTD) of RNA Polymerase II (Pol II) that provide access to the type and position of modifications of a LC domain by mass spectrometry (MS).

DEAD-box helicase DDX27 regulates 3' end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex.

PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3 end formation of 47S rRNA independently of the PeBoW-complex.

Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3 direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.

Transcriptome analysis of dominant-negative Brd4 mutants identifies Brd4-specific target genes of small molecule inhibitor JQ1

The bromodomain protein Brd4 is an epigenetic reader and plays a critical role in the development and maintenance of leukemia. Brd4 binds to acetylated histone tails and activates transcription by recruiting the positive elongation factor P-TEFb. Small molecule inhibitor JQ1 competitively binds the bromodomains of Brd4 and displaces the protein from acetylated histones. However, it remains unclear whether genes targeted by JQ1 are mainly regulated by Brd4 or by other bromodomain proteins such as Brd2 and Brd3. Here, we describe anti-proliferative dominant-negative Brd4 mutants that compete with the function of distinct Brd4 domains. We used these Brd4 mutants to compare the Brd4-specific transcriptome with the transcriptome of JQ1-treated cells. We found that most JQ1-regulated genes are also regulated by dominant-negative Brd4 mutants, including the mutant that competes with the P-TEFb recruitment function of Brd4. Importantly, JQ1 and dominant-negative Brd4 mutants regulated the same set of target genes of c-Myc, a key regulator of the JQ1 response in leukemia cells. Our results suggest that Brd4 mediates most of the anti-cancer effects of JQ1 and that the major function of Brd4 in this process is the recruitment of P-TEFb. In summary, our studies define the molecular targets of JQ1 in more detail.

A Nonradioactive Assay to Measure Production and Processing of Ribosomal RNA by 4sU-Tagging

In vivo metabolic pulse labeling is a classical approach to assess production and processing of ribosomal RNA (rRNA). However, conventional labeling techniques can be indirect and require work with radioactivity. Here, we describe in detail a protocol for in vivo metabolic labeling, purification, and readout of nascent rRNA by 4-thiouridine (4sU). We propose 4sU labeling as standard nonradioactive technique for the analysis of rRNA metabolism during ribosome biogenesis.