RNA polymerase II elongation control

Abstract

Regulation of the elongation phase of transcription by RNA polymerase II (Pol II) is utilized extensively to generate the pattern of mRNAs needed to specify cell types and to respond to environmental changes. After Pol II initiates, negative elongation factors cause it to pause in a promoter proximal position. These polymerases are poised to respond to the positive transcription elongation factor P-TEFb, and then enter productive elongation only under the appropriate set of signals to generate full-length properly processed mRNAs. Recent global analyses of Pol II and elongation factors, mechanisms that regulate P-TEFb involving the 7SK small nuclear ribonucleoprotein (snRNP), factors that control both the negative and positive elongation properties of Pol II, and the mRNA processing events that are coupled with elongation are discussed.

Figure 1. Divergent transcription, promoter proximally paused polymerases, and productive elongation

Many promoters (purple box) allow Pol II to initiate and elongate in both directions using two transcription start sites. Transcription upstream of the gene gives rise to short unstable transcripts and transcription into the gene produces paused polymerases that are poised for entry into productive elongation or termination. The thickness of the arrows is represent relative flow of polymerases; initiation (black), termination (pink), transition into productive elongation (green).

Figure 2. Pol II and elongation factor occupancy from ChIP-Seq

(A) MES cell ChIP-Seq datasets from Rahl et al. 2010 (9) were used to generate the views of Pol II from control or Flavopiridol treated cells as well as Ser5 and Ser2 phosphorylation of the CTD of the large subunit of Pol II across two neighboring genes on mouse chromosome 4. The data for each track for each gene was normalized so that the area under all curves was equal (except for the Ser2P for TCEB3 for which there was no data). (B) Metagene analysis of MES cell ChIP-Seq data (9) for Pol II, Spt5 subunit of DSIF and NELFe subunit of NELF. The relative distributions of the polymerase and factors were compiled for the region from −10,000 to +10,000 bp around the TSSs for ~20,000 RefSeq genes. In (A) and (B) background signals from the lowest 10% of the 20,000 data points in each distribution were averaged and subtracted from all data points and the resulting curves were normalized so that the total area under each was equal. (C) Diagram of the engaged polymerase with nascent transcript (red line) under the influence of DSIF and NELF.

Figure 3. Regulation of P-TEFb by the 7SK snRNP

7SK snRNA is depicted in a cartoon view in which the secondary structures known to be involved in its function are shown. The LA Related Protein, LARP7, is constitutively associated with 7SK and the Methyl Phosphate Capping enzyme, MePCE, which methylates the triphosphate at the 5’ end of 7SK is also bound. After binding to the major 5’ stem and loop HEXIM1 or HEXIM2 (HEXIM) undergoes a conformational change and binds to and inhibits P-TEFb. When P-TEFb is released from the 7SK snRNP, HEXIM is also released and there is a structural change in 7SK. hnRNPs then replace P-TEFb and HEXIM.

Figure 4. P-TEFb mediated transition into productive elongation

Regulation of HIV transcription is used as an example of how P-TEFb is released from the 7SK snRNP and specifically recruited to its site of action. HIV Tat interacts with P-TEFb in the 7SK snRNP leading to extraction of P-TEFb and the formation of a Tat P-TEFb complex. P-TEFb is recruited to the poised polymerase on the HIV LTR by an interaction with the HIV nascent transcript TAR, where it phosphorylates DSIF (P) as well as NELF and the polymerase (not shown). Also not shown is the SEC which can accompany and potentially help recruit P-TEFb.

Figure 5. The super elongation complex, SEC

In the absence of sequence-specific recruitment factors such as HIV-1 Tat and the MLL fusions, the SEC complex, which is assembled around the scaffolding protein AFF4, is recruited to the elongating Pol II through the interaction of the YEATS domain of either ENL or AF9 with the PAF1 subunit of PAFc. This allows the SEC to use its P-TEFb and ELL2 functional modules to exert a multitude of effects (e.g. direct stimulation of the Pol II catalytic rate by ELL2, phosphorylation of Ser2 on the Pol II CTD and DSIF by P-TEFb, and phosphorylation and release of NELF by P-TEFb) that result in the synergistic activation of Pol II elongation.

Figure 6. Coupling of 3' end processing with transcription

Polyadenylation factors such as CstF, CPSF and symplekin are recruited to the Pol II elongation complex through the concerted actions of the phospho-Ser2 CTD and transcription elongation factors ELL2 and PAFc, which track along with Pol II during productive elongation. Once the cleavage/polyadenylation signals (AAUAAA followed by a G/U-rich sequence) emerge in the nascent mRNA, they are recognized by the processing machinery to result in efficient polyadenylation.