The ribonucleotidyl transferase USIP-1 acts with SART3 to promote U6 snRNA recycling

Abstract

The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1-U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling.

Figure 1.

B0035.12 encodes an essential, nuclear protein orthologous to SART3. (A) Schematic representation of the domain structure of Prp24p from Saccharomyces cerevisiae, SART3 from Homo sapiens, and SART3 from Caenorhabditis elegans. xe3 denotes an N-terminal truncation allele of SART3. Numbers indicate amino acid positions. HAT = half a TPR, RRM = RNA recognition motif, LID = Lsm interaction domain. (B) Western blot of lysates extracted from L4 stage worms. Transgenic and endogenous SART3 were detected with an affinity-purified polyclonal antibody against SART3 in lysates of worms homozygous for the xe3 allele (lane 1), which we obtained by sorting, wild-type worms (lane 2), and xe3 homozygous mutant worms rescued by transgenic sart-3 containing a C-terminal GFP/His/FLAG-tag (GHF) (lane 3). (C) Differential interference contrast (DIC) micrographs. Adult worms homozygous for the xe3 allele burst through the vulva (left panel). By contrast, xe3 worms expressing transgenic sart-3::gfp::his::flag (middle panel), like wild-type worms (right panel), do not burst. Arrows point to the vulva. Scale bar, 20 μm. (D) Fluorescence and DIC micrographs of L4 stage worms expressing C-terminally GFP/3xFLAG-tagged SART3 from a fosmid. Pharynx signal in the head (top left panel) arises from an RFP co-injection marker leaking into the GFP channel. Arrows point to nucleoplasm, arrow heads point to nucleolus. Scale bar, 20 μm. (E) Western blot with lysates from wild-type worms extracted at different time points during development. L1 = larval stage 1, etc.

Figure 2.

SART3 co-immunoprecipitates U4 and U6 snRNPs. (A) Western blot of anti-FLAG co-immunoprecipitation (co-IP) of N-terminally FLAG-tagged SART3 (lanes 1–3) and a GFP/His/FLAG control construct (lanes 4–6). Seven percent of input and 15% of IPs were loaded on a gel. (B) Mass spectrometry results of FLAG/SART3 co-IPs. Numbers of spectra mapping uniquely to a given protein are indicated. IP1: protein extraction using dounce homogenizer, RNase A treatment performed at room temperature, eluate concentration by TCA precipitation. IP2: protein extraction using mortar and pestle, RNase A treatment performed at 4°C, eluate concentration by speed vac. GUT-2 is homologous to human Lsm2 and the SNR proteins to Sm proteins. The percentage coverage for these proteins is provided in Supplementary Table S1. (C) Northern blot of RNA extracted from eluates obtained by an anti-FLAG co-IP on lysates from worms expressing the indicated transgene. Two milligram total protein was used as input for the co-IP and 100% of the co-immunoprecipitated RNA was loaded on the gel. 2.5 μg of total RNA were loaded as a reference. Subsets of probes were applied to the membrane to detect different RNAs simultaneously; the asterisk indicates an unspecific band detected with the probe against U6.

Figure 3.

SART3 and USIP1/ZK863.4 are in a U6 snRNA-containing complex. (A) Western blot of anti-FLAG co-IP of C-terminally GFP/3xFLAG-tagged USIP-1 (lanes 1 and 3–5) and a C-terminally His/FLAG-tagged GFP control construct (lanes 2 and 6–8). USIP-1/GFP/3xFLAG and GFP/His/FLAG constructs were detected with anti-GFP. Endogenous SART3 was detected with a polyclonal antibody against SART3. Two percent of input and 70% of IPs were loaded on a gel. (B) Proteins identified by mass spectrometry following IP of FLAG-tagged SART3, USIP-1 or GFP. The latter serves as a negative control (see additional proteins in Supplementary Table S2). Numbers of spectra mapping uniquely to a given protein are indicated. (C) Northern blot of RNA extracted from eluates obtained by an anti-FLAG co-IP on lysates from worms expressing the indicated transgene. Two milligram total protein was used as input for the co-IP and 100% of the co-immunoprecipitated RNA was loaded on the gel. 2.5 μg of total RNA were loaded as a reference. Subsets of probes were applied to the membrane to detect different RNAs simultaneously; the asterisk indicates an unspecific band detected with the probe against U6. (D) Fluorescence and DIC microscopy of L4 stage worms expressing C-terminally GFP/3xFLAG-tagged USIP-1 from a fosmid. Arrows point to nucleoplasm, arrow heads point to nucleolus. Scale bar, 20 μm.

Figure 4.

USIP-1 is a terminal uridylyl transferase. (A) Schematic representation of the domain structure of STPAP/RBM21/TUT1 (UniProt: STPAP_HUMAN) and MTPAP/PAPD1 (PAPD1_HUMAN) from H. sapiens, and the C. elegans proteins GLD-4 (GLD4_CAEEL) and USIP-1 (Q23652_CAEEL). tm1897 denotes a deletion allele of USIP-1 that leads to a frame shift (striated) and premature termination codon at position 276. C₂H₂ ZF = C₂H₂-type (classical) zinc finger (ZF), RRM = RNA recognition motif, PAD = PAP-associated domain, DXD: Amino acid motif required for catalytic activity where D = aspartic acid and X = any amino acid. (B) Assay to test terminal transferase activity of recombinant USIP-1 and USIP-1^cd. Twenty micrograms of recombinant protein was added to 200 ng of a synthetic 22-nucleotide-long RNA substrate in the presence of 1 mM UTP. The reaction was stopped by addition of Trizol between 0 and 10 min. Shown is a SYBR Gold staining of a 15% urea–polyacrylamide gel (inverted picture). M = Marker. (C) Transferase assay similar to (B) with different nucleotide triphosphates. (D) Autoradiography of transferase activity of recombinant USIP-1 or USIP-1^cd on gel-purified U6 RNA from the indicated sources using radioactively labeled UTP-α-^[32]P.

Figure 5.

Synthetic embryonic lethality occurs when simultaneously compromising SART3 and USIP-1 activity. (A) Wild-type worms or worms homozygous for the tm1897 allele were exposed to mock or sart-3 RNAi at the L1 stage (P0 generation) and cultured at 25°C. After 60 h, the hatched progeny (F1 generation) was counted. n = 3, error bars indicate SEM. (B) Wild-type worms or worms homozygous for the tm1897 allele were exposed to mock or sart-3 RNAi at the L1 stage (P0 generation) and cultured at 25°C. Pictures of the next generation (F1 generation) were taken after 96 h revealing hatched F1's (that were arrested due to lack of food though) for wild-type; mock RNAi, wild-type; sart-3 RNAi and usip-1(tm1897); mock RNAi but arrested embryos or L1 stage worms (arrow) for usip-1(tm1897); sart-3 RNAi. (C) Western blotting shows similar protein levels for transgenic, FLAG-tagged wild-type USIP-1 or mutant USIP-1^cd. Both transgenes are expressed in a usip-1(tm1897) background and were detected by an anti-FLAG antibody.

Figure 6.

USIP-1 stabilizes U6 snRNA. (A) Northern blot with RNA extracted from wild-type worms (N2) or usip-1(tm1897) worms. The latter contain either no transgene or transgenic usip-1 or usip-1^cd. (B) Quantification of northern blots as shown in (A) from experiments with four biological replicates, mean + SEM. *P-value < 0.05 by a paired two-tailed t-test; n.s., not statistically significant. (C) Northern blot with RNA isolated from immunoprecipitated FLAG/SART3. FLAG/SART3 was immunoprecipitated from wild-type worms (usip-1(+)) or usip-1(tm1897) worms. Two replicates are shown. Quantification of bands is relative to the underlined value, which has been set to 1. (D) 3′ end sequences of U6 snRNA from wild-type and usip-1(tm1897) worms were determined by 3′ RACE analysis and grouped as indicated according to length of the oligo-U tail. [Note that only tail sequences consisting entirely of Us were used for the analysis depicted here, but that comparable results were obtained when including the occasional tail sequences that contain As in addition to Us (data not shown).]

Figure 7.

Model of the life cycle of U6 snRNA. Additional snRNP-associated proteins (1) are omitted for the sake of simplicity. See text for details.