Karel Kubicek

Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

Molecular Basis for Coordinating Transcription Termination with Noncoding RNA Degradation

Summary The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

In vivo SELEX reveals novel sequence and structural determinants of Nrd1-Nab3-Sen1-dependent transcription termination.

The Nrd1‐Nab3‐Sen1 (NNS) complex pathway is responsible for transcription termination of cryptic unstable transcripts and sn/snoRNAs. The NNS complex recognizes short motifs on the nascent RNA, but the presence of these sequences alone is not sufficient to define a functional terminator. We generated a homogeneous set of several hundreds of artificial, NNS‐dependent terminators with an in vivo selection approach. Analysis of these terminators revealed novel and extended sequence determinants for transcription termination and NNS complex binding as well as supermotifs that are critical for termination. Biochemical and structural data revealed that affinity and specificity of RNA recognition by Nab3p relies on induced fit recognition implicating an α‐helical extension of the RNA recognition motif. Interestingly, the same motifs can be recognized by the NNS or the mRNA termination complex depending on their position relative to the start of transcription, suggesting that they function as general transcriptional insulators to prevent interference between the non‐coding and the coding yeast transcriptomes.

Recognition of Transcription Termination Signal by the Nuclear Polyadenylated RNA-binding (NAB) 3 Protein

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

Recognition of asymmetrically dimethylated arginine by TDRD3

Asymmetric dimethylarginine (aDMA) marks are placed on histones and the C-terminal domain (CTD) of RNA Polymerase II (RNAP II) and serve as a signal for recruitment of appropriate transcription and processing factors in coordination with transcription cycle. In contrast to other Tudor domain-containing proteins, Tudor domain-containing protein 3 (TDRD3) associates selectively with the aDMA marks but not with other methylarginine motifs. Here, we report the solution structure of the Tudor domain of TDRD3 bound to the asymmetrically dimethylated CTD. The structure and mutational analysis provide a molecular basis for how TDRD3 recognizes the aDMA mark. The unique aromatic cavity of the TDRD3 Tudor domain with a tyrosine in position 566 creates a selectivity filter for the aDMA residue. Our work contributes to the understanding of substrate selectivity rules of the Tudor aromatic cavity, which is an important structural motif for reading of methylation marks.

The Tubulin-Bound Structure of the Antimitotic Drug Tubulysin

Bound to be active: The solution structure of tubulin-bound tubulysin A is determined from transferred NOE data (see picture; blue N, red O, yellow S, green C), and this bioactive conformation is compared to the unbound conformation. The binding site on tubulin is examined on the basis of the interligand NOEs observed between epothilone A and tubulysin A in the presence of tubulin.

A further investigation of the cytochrome b5–cytochrome c complex

The interaction of reduced rabbit cytochrome b5 with reduced yeast iso-1 cytochrome c has been studied through the analysis of 1H–15N HSQC spectra, of 15N longitudinal (R1) and transverse (R2) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b5 has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b5.

Platinum–DNA interstrand crosslinks: Molecular determinants of bending and unwinding of the double helix

Platinum diamine complexes are able to crosslink the guanines of d(GC)(2) dinucleotides within double-stranded DNA. The interstrand crosslink thus formed causes a bend of the double helix toward the minor groove and the helical sense changes locally to left-handed, resulting in a considerable unwinding. The bend and unwinding angles have been shown to depend on the platinum ligands. Here, we have used molecular dynamics simulations to investigate the DNA 20-mer d(C(1)T(2)C(3)T(4)C(5)C(6)T(7)T(8)G*(9)C(10)T(11)C(12)T(13)C(14)C(15)T(16)T(17)C(18)T(19)C(20))-d(G(21)A(22)G(23)A(24)A(25)G(26)G(27)A(28)G(29)A(30)G*(31)C(32)A(33)A(34)G(35)G(36)A(37)G(38)A(39)G(40)) with the G* guanines crosslinked by cis-Pt(NH(3))(2)(2+), Pt(R,R-DACH)(2+), or Pt(S,S-DACH)(2+). Previous investigations on cisplatin interstrand adducts indicated that the structure is similar in solid state and in solution; thus, we used the reported X-ray structure of a cisplatin adduct as a starting model. Replacing in the MD-relaxed model for the DNA duplex crosslinked with cis-Pt(NH(3))(2)(2+) the two NH(3) platinum ligands by R,R-DACH or S,S-DACH led to clashes between the DACH residue and the deoxyribose of C(12). Confrontation of MD-derived models with gel shift measurements suggested that these clashes are avoided differently in the adducts of Pt(R,R-DACH)(2+)versus Pt(S,S-DACH)(2+). The R,R-isomer avoids the clash by untwisting the T(11)/A(30)-C(12)/G(29) step, thus increasing the global unwinding. In contrast, the S,S-isomer modifies the shift and slide parameters of this step, which dislocates the helical axis and enhances the bend angle. The clash that leads to the differentiation of the structures as a function of the diamine ligand is related to a hydrogen bond between the platinum complex and the T(11) base and could be characteristic of interstrand crosslinks at d(pyG*Cpy)-d(puG*Cpu) sequences.

Structural insight into recognition of phosphorylated threonine‐4 of RNA polymerase II C‐terminal domain by Rtt103p

Phosphorylation patterns of the C‐terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD‐interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho‐Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103ps CID binds equally well Thr4 and Ser2 phosphorylated CTD. A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID‐containing CTD‐binding factor may be coded by more than one letter of the CTD code.

Structure and semi-sequence-specific RNA binding of Nrd1.

In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix–loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix–loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.