Hyperphosphorylated C-terminal Repeat Domain-associating Proteins in the Nuclear Proteome Link Transcription to DNA/Chromatin Modification and RNA Processing

Abstract

Using an interaction blot approach to search in the human nuclear proteome, we identified eight novel proteins that bind the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of RNA polymerase II. Unexpectedly, five of the new phosphoCTD-associating proteins (PCAPs) represent either enzymes that act on DNA and chromatin (topoisomerase I, DNA (cytosine-5) methyltransferase 1, poly(ADP-ribose) polymerase-1) or proteins known to bind DNA (heterogeneous nuclear ribonucleoprotein (hnRNP) U/SAF-A, hnRNP D). The other three PCAPs represent factors involved in pre-mRNA metabolism as anticipated (CA150, NSAP1/hnRNP Q, hnRNP R) (note that hnRNP U/SAF-A and hnRNP D are also implicated in pre-mRNA metabolism). Identifying as PCAPs proteins involved in diverse DNA transactions suggests that the range of phosphoCTD functions extends far beyond just transcription and RNA processing. In view of the activities possessed by the DNA-directed PCAPs, it is likely that the phosphoCTD plays important roles in genome integrity, epigenetic regulation, and potentially nuclear structure. We present a model in which the phosphoCTD association of the PCAPs poises them to act either on the nascent transcript or on the DNA/chromatin template. We propose that the phosphoCTD of elongating RNA polymerase II is a major organizer of nuclear functions. Molecular & Cellular Proteomics 1:598 -610, 2002.The C-terminal repeat domain (CTD), 1 a non-catalytic "tail" on the largest subunit of RNA polymerase, is a unique structure made up of as many as 52 repeats of the consensus heptamer YSPTSPS (1). That each seven-residue repeat contains five hydroxyl-containing side chains suggests that the CTD could be a good target for phosphorylation, and indeed, in elongating RNAP II the CTD is hyperphosphorylated (2-4); in preinitiating RNAP II, on the other hand, the CTD is not phosphorylated (e.g. see Ref. 5). The amino acid composition of the CTD also renders it very hydrophilic, and it is largely unstructured in aqueous solution (6, 7); in mammals a fully stretched out CTD could potentially extend some 1200 Å away from the globular body of the polymerase (whose diameter is ϳ150 Å, Ref. 8) (see e.g. Ref. 9). Its unusual properties render the CTD well suited to function as an extended scaffold to which transcription factors and other proteins could bind; indeed, a growing body of data indicates that such a scaffolding role is the principal function of the CTD. Experiments focusing on the hyperphosphorylated CTD characteristic of elongating RNAP II have revealed that a number of RNA processing factors bind to the phosphoCTD (e.g. see Refs. 10 and 11). Binding to the phosphoCTD tethers such factors to the transcription machinery where they can carry out their functions more efficiently (e.g. see Refs. 12-16); furthermore, certain factors or processes are stimulated by phosphoCTD binding (e.g. see . In addition, a pool of nontranscribing RNAP II carries a phosphoCTD and is associated with certain transcription and processing factors in potential assembly ar...

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…This ionic strength [0.4 M (NH 4 ) 2 SO 4 ] has been shown to be sufficient to extract factors associated with elongating Pol II from yeast (42,43), and we have found that it is also more than sufficient to disengage virtually all mammalian PCAPs from RNA Pol II in a chromatin pellet from HeLa cells (39). The crude extract was centrifuged at 14000g (8000 rpm in a Sorvall SLC-6000 rotor) for 45 min at 4 °C to remove cell debris.…”

“…For example, fractions 23, 25, and 27 have several bands that are detected by the PCTD probe. The pattern of possible phosphoCTD-interacting proteins does not mirror the profile of proteins eluted from the ion-exchange column (not all lanes have putative PCAPs, and not all proteins in any given lane interact with the probe), arguing against the simple possibility that the PCTD probe acts merely as a polyanion (see also discussion in ref 39 and Figure 5G-J).…”

“…For example, CTD phosphorylation by CTDK-I also plays a role in recruiting the histone H3 Lys36 methyltransferase Set2, and ctk1Δ cells are defective for histone H3 Lys36 methylation (37,38). Moreover, five of eight mammalian phosphoCTD-associating proteins (PCAPs) recently identified play roles in DNA or chromatin transactions (39). These and other results suggest that the functional repertoires of both the PCTD and its kinases have been greatly underestimated.…”

RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability

“…TOP1 is an essential enzyme in mammals 30 , and one of its primary functions during cell growth is to maintain DNA topology by relaxing supercoiled DNA 6 . TOP1 can localize to actively transcribed regions and associate with the RNAPIIo elongation complex in vertebrates 31,32 . Our analysis provides insight into the biological function of the TOP1 K391/K436 SUMO modifications during transcription.…”

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb

“…The transition from pausing to elongation is facilitated by the P-TEFb elongation complex, which also mediates efficient elongation (10). P-TEFb consists of two subunits, cyclin T1 and the kinase CDK9, which phosphorylates serine 2 of the CTD, required for productive elongation and the recruitment of complexes involved in mRNA processing (splicing and polyadenylation) (10)(11)(12)(13)(14)(15).…”