IT'S 2 for the price of 1: Multifaceted ITS2 processing machines in RNA and DNA maintenance

Abstract

Cells utilize sophisticated RNA processing machines to ensure the quality of RNA. Many RNA processing machines have been further implicated in regulating the DNA damage response signifying a strong link between RNA processing and genome maintenance. One of the most intricate and highly regulated RNA processing pathways is the processing of the precursor ribosomal RNA (pre-rRNA), which is paramount for the production of ribosomes. Removal of the Internal Transcribed Spacer 2 (ITS2), located between the 5.8S and 25S rRNA, is one of the most complex steps of ribosome assembly. Processing of the ITS2 is initiated by the newly discovered endoribonuclease Las1, which cleaves at the C2 site within the ITS2, generating products that are further processed by the polynucleotide kinase Grc3, the 5'→3' exonuclease Rat1, and the 3'→5' RNA exosome complex. In addition to their defined roles in ITS2 processing, these critical cellular machines participate in other stages of ribosome assembly, turnover of numerous cellular RNAs, and genome maintenance. Here we summarize recent work defining the molecular mechanisms of ITS2 processing by these essential RNA processing machines and highlight their emerging roles in transcription termination, heterochromatin function, telomere maintenance, and DNA repair.

Keywords: Endoribonuclease; Exonuclease; Genome maintenance; Grc3; Las1; Polynucleotide kinase; RNA exosome; Rat1/Xrn2; rRNA processing.

Fig. 1.

(A) Cartoon of the S. cerevisiae pre-rRNA processing pathway. The 35S prerRNA is transcribed by Pol I and includes the 5′ ETS (external transcribed spacer), the 18S rRNA, the ITS1 (internal transcribed spacer), the 5.8S rRNA, the ITS2, the 25S rRNA, and the 3′ ETS. Letters above annotate known cleavage sites (15,28,29,164). Processing of the ITS2 is initiated by the Las1-Grc3 complex (blue), composed of the Las1 nuclease and Grc3 kinase, by cleavage at the C2 site. This generates the 7S pre-rRNA with a 2′−3′-cyclic phosphate (2′−3′ cP) and the 26S prerRNA with a 5′-hydroxyl (5′-OH). Following C2 cleavage by Las1, Grc3 phosphorylates the 5′-hydroxyl end of the 26S pre-rRNA, which targets the 26S pre-rRNA for degradation by the 5′→3′ exonuclease Rat1 (purple). The 3′-end of the 7S pre-rRNA is then subsequently processed by the 3′→5′ RNA exosome (gray/red). (B) Secondary structure of the ring-pin ITS2 model. This model was adapted from (23) and is based on RNA structure probing experiments.

Fig. 2.

Structural features of the Rat1/Xrn2 5′→3′ exoribonuclease. (A) Boundaries of the Rat1/Xrn2 and Xrn1 conserved N-terminal catalytic exoribonuclease domain (dark purple) and divergent C-terminus (light purple). Numbers above the bar diagram represent the amino acid residues. Abbreviations are as follows: Schizosaccharomyces pombe (Sp), Caenorhabditis elegans (Ce) and Drosophila melanogaster (Dm). (B) Structures of the conserved catalytic domain from S. pombe Rat1 (left) bound to its cofactor Rai1 in light green, C. elegans Xrn2 (middle) associated with its cofactor PAXT-1 shown in dark green and D. melanogaster Xrn1 (right) bound to a 5′-monophosphate trinucleotide substrate colored in beige (5′-P oligo). Black boxes mark the narrow entrance of the exoribonuclease active site. The insets are zooms of the exoribonuclease active site where invariant catalytic residues are labeled, a magnesium ion is shown as a gray sphere and black dotted lines represent hydrogen bonds. DmXrn1 basic patch residues (R100, R101) are responsible for interacting with the 5′-monophosphate (5′-P) while the RNA clamp residues (H41, W540) π- π stack with the incoming RNA substrate aligning the scissile phosphate (asterisk) with catalytic residues (D35, E177, D205) for RNA cleavage. These critical DmXrn1 residues are conserved in SpRat1 and CeXrn2 suggesting Rat1/Xrn2 undergoes a similar catalytic mechanism. Coordinates are from the following: S. pombe Rat1-Rai1 PDB ID 3FQD, C. elegans Xrn2-PAXT-1 PDB ID 5FIR and D. melanogaster substrate-engaged Xrn1 PDB ID 2Y35.

Fig. 3.

Pre-rRNA processing by the RNA exosome complex. (A) The multisubunit RNA exosome relies on cofactors and adapters for substrate recruitment. Together the barrel-shaped inactive Exo-9 core (gray) assembles with exonucleases Rrp44 (light red) and Rrp6 (dark red), and the cofactor Rrp47 (yellow) to form the Exo-12 complex. In the closed conformation, Rrp6 occupies the Mtr4 binding region of Exo-9 which blocks the recruitment of Mtr4 and prevents RNA from passing through the central channel. (B) Association of Mpp6 (purple) and the transiently associated Mtr4 (orange) forms the Exo-14 complex. Exo-14 undergoes a conformational change upon engagement with pre-60S particles for ITS2 processing. This conformational change opens the central channel and creates a direct path for the ITS2 from the Mtr4 helicase, through the central channel to the Rrp44 exonuclease active site. The coordinates for the pre-60S were taken from PDB ID 6FTS.

Fig. 4.

Non-canonical activities of ITS2 processing factors. DNA/RNA hybrids are found at sites of diverse DNA transactions including DNA transcription termination, heterochromatic gene silencing, telomere maintenance and transcription-associated DNA double strand breaks (DSBs). (A) The Rat1 5′→3′ exoribonuclease degrades the RNA polymerase-associated RNA transcript to terminate DNA transcription. (B) The RNA exosome and Rat1 exoribonucleases degrade pre-mRNA transcripts produced at heterochromatin for post-transcriptional gene silencing. (C) Rat1 targets telomeric repeat-containing RNA (TERRA) produced at telomeres to prevent the accumulation of DNA/RNA hybrids that deter the progression of Telomerase. (D) Xrn2 is found at RNA polymerase pause sites for transcription-associated DNA double strand break (DBS) repair. Xrn2 associates with DNA repair factor 53BP1 to signal for the recruitment of nonhomologous end joining (NHEJ) factors to orchestrate a DNA damage response.