RBM7 subunit of the NEXT complex binds U-rich sequences and targets 3'-end extended forms of snRNAs.

Stepanka Vanacova,Marek Bartosovic,David Potesil,Dominika Hrossova,Josef Pasulka,Richard Stefl,Zbynek Zdrahal,Tomas Sikorsky

doi:10.1093/nar/gkv240

Abstract

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

Highlights

The recent expansion of transcriptome-wide screens led to the identification of the complexity of the eukaryotic non-coding RNAome
We have recently identified several proteins that were precipitated on oligo(U) RNA from mouse embryonic stem cell extracts [24]
Part of the nucleoplasmic proteins was detected in the cytoplasmic fractions, which was probably due to the partial leakage of the nucleoplasm during the fractionation (Figure 1B)

Summary

Introduction

The TRAMP is composed of the non-canonical poly(A)polymerase (PAP) Trf4p or Trf5p, the zinc-knuckle (ZnK) protein Air1p or Air2p and the RNA helicase Mtr4p [4,5,6,7]. The NNS is formed by two RNA-binding proteins Nrd1p and Nab3p and an RNA helicase Sen1p [8]. It is believed, that the NNS complex has a central role in RNA polymerase II (RNAP II) transcription termination of noncoding RNAs such as cryptic unstable transcripts (CUTs), or sn/snoRNAs [9,10,11]. After termination the NNS complex interacts with the TRAMP-exosome and mediates 3 end trimming and maturation or complete degradation of a substrate [3,11,15]

Methods

Results

Conclusion