Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA

Kaspar Burger,Bastian Mühl,Michaela Rohrmoser,Britta Coordes,Martin Heidemann,Markus Kellner,Anita Gruber-Eber,Vigo Heissmeyer,Katja Strässer,Dirk Eick

doi:10.1074/jbc.m113.483719

Abstract

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

Highlights

Processing of ribosomal RNA is sensitive to cyclin-dependent kinases (Cdks) inhibitors
U2OS cells were transfected with siRNA targeting mRNAs of Cdk1 to Cdk16 or with control siRNA for mRNAs of luciferase, CATS, and the ribosomal RNA (rRNA) processing factor Pescadillo 1 (Pes1) (29, 30) (Fig. 1, supplemental Fig. S2)
cyclin-dependent kinase 9 (Cdk9)-mediated RNA polymerase II (RNAPII) Transcription Is Required for 47 S rRNA Processing—Processing of rRNA is highly sensitive to Cdk inhibition by FL

Summary

Background

Processing of ribosomal RNA is sensitive to Cdk inhibitors. Results: Inhibition of Cdk activity blocks 47 S rRNA processing and stabilizes a 3Ј extended 47 S primary transcript. Our data demonstrate a pivotal role of Cdk activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing. The 47 S transcript is subject to extensive co-transcriptional modification by methylation and pseudouridylation and a cascade of endo- and exonucleolytic cleavage and degradation steps These steps are tightly connected with the association and dissociation of processing factors to and from pre-ribosomes and result in the production of mature 40 S and 60 S ribosomal subunits (1–3). Our data suggest a model in which inhibition of Cdk prevents the proper production of pre-mRNA-embedded snoRNAs and thereby inhibits rRNA processing. This model links RNAPII activity directly to ribosome biogenesis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION