ABSTRACT
The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.
Subject(s)
Carrier Proteins/physiology , Models, Biological , Nuclear Proteins/physiology , RNA Helicases/physiology , RNA-Binding Proteins/physiology , Ribonucleases/metabolism , Carrier Proteins/analysis , Carrier Proteins/metabolism , Cell Nucleolus/enzymology , Cell Nucleolus/metabolism , Chromosomal Proteins, Non-Histone/analysis , Chromosomal Proteins, Non-Histone/metabolism , DNA-Directed DNA Polymerase/analysis , DNA-Directed DNA Polymerase/metabolism , Exoribonucleases/analysis , Exoribonucleases/metabolism , Exoribonucleases/physiology , Exosome Multienzyme Ribonuclease Complex , Humans , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , RNA Helicases/analysis , RNA Helicases/metabolism , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Transcription Factors/analysis , Transcription Factors/metabolismABSTRACT
PROMoter uPstream Transcripts (PROMPTs) were identified as a new class of human RNAs, which are heterologous in length and produced only upstream of the promoters of active protein-coding genes. Here, we show that PROMPTs carry 3'-adenosine tails and 5'-cap structures. However, unlike mRNAs, PROMPTs are largely nuclear and rapidly turned over by the RNA exosome. PROMPT-transcribing DNA is occupied by RNA polymerase II (RNAPII) complexes with serine 2 phosphorylated C-terminal domains (CTDs), mimicking that of the associated genic region. Thus, the inefficient elongation capacity of PROMPT transcription cannot solely be assigned to poor CTD phosphorylation. Conditions that reduce gene transcription increase RNAPII occupancy of the upstream PROMPT region, suggesting that they reside in a common transcription compartment. Surprisingly, gene promoters that are actively transcribed by RNAPI or RNAPIII also produce PROMPTs that are targeted by the exosome. RNAPIII PROMPTs bear hallmarks of RNAPII promoter-associated RNAs, explaining the physical presence of RNAPII upstream of many RNAPIII-transcribed genes. We propose that RNAPII activity upstream gene promoters are wide-spread and integral to the act of gene transcription.
Subject(s)
Promoter Regions, Genetic , RNA, Nuclear/chemistry , Cyclin D1/genetics , Genes, myc , HEK293 Cells , HeLa Cells , Humans , Polyadenylation , RNA Nucleotidyltransferases/metabolism , RNA Polymerase I/metabolism , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Polymerase III/metabolism , RNA, Messenger/chemistry , RNA, Nuclear/metabolism , Serine/metabolismABSTRACT
Studies have shown that the bulk of eukaryotic genomes is transcribed. Transcriptome maps are frequently updated, but low-abundant transcripts have probably gone unnoticed. To eliminate RNA degradation, we depleted the exonucleolytic RNA exosome from human cells and then subjected the RNA to tiling microarray analysis. This revealed a class of short, polyadenylated and highly unstable RNAs. These promoter upstream transcripts (PROMPTs) are produced approximately 0.5 to 2.5 kilobases upstream of active transcription start sites. PROMPT transcription occurs in both sense and antisense directions with respect to the downstream gene. In addition, it requires the presence of the gene promoter and is positively correlated with gene activity. We propose that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential.
Subject(s)
Exosomes/metabolism , Promoter Regions, Genetic , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic , DNA Methylation , HeLa Cells , Humans , Oligonucleotide Array Sequence Analysis , RNA Polymerase II/metabolism , RNA Stability , Transcription Factors/metabolism , Transcription Initiation Site , TransfectionABSTRACT
Heat shock genes respond to moderate heat stress by a wave of transcription. The induction phase is accompanied by the massive eviction of histones, which later reassemble with DNA during the ensuing phase of transcription downregulation. In this article, we identify determinants of this reassembly throughout the heat shock protein 104 gene (HSP104) transcription unit. The results show that, although histone H3 lacking amino acids 4-30 of its N-terminal tail (H3Delta4-30) is normally deposited, reassembly of H3Delta4-40 is obliterated with an accompanying sustained transcription. On mutation of the histone chaperones Spt6p and Spt16p, but not Asf1p, reassociation of H3 with DNA is compromised. However, despite a lasting open chromatin structure, transcription ceases normally in the spt6 mutant. Thus, transcriptional downregulation can be uncoupled from histone redeposition and ongoing transcription is not required to prevent chromatin reassembly.
