A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours.

Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.

SMUG1 Promotes Telomere Maintenance through Telomerase RNA Processing.

Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3'-polyadenylated and 3'-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1.

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/- mice. We found that 5-hydroxymethyluracil accumulated in Smug1 -/- tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 -/- mice did not accumulate uracil in their genome and Ung -/- mice showed slightly elevated uracil levels. Contrastingly, Ung -/- Smug1 -/- mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

The human base excision repair enzyme SMUG1 directly interacts with DKC1 and contributes to RNA quality control.

Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that removes uracil and oxidised pyrimidines from DNA. We show that SMUG1 interacts with the pseudouridine synthase Dyskerin (DKC1) and colocalizes with DKC1 in nucleoli and Cajal bodies. As DKC1 functions in RNA processing, we tested whether SMUG1 excised modified bases in RNA and demonstrated that SMUG1 has activity on single-stranded RNA containing 5-hydroxymethyldeoxyuridine, but not pseudouridine, the nucleoside resulting from isomerization of uridine by DKC1. Moreover, SMUG1 associates with the 47S rRNA precursor processed by DKC1, and depletion of SMUG1 leads to a reduction in the levels of mature rRNA accompanied by an increase in polyadenylated rRNA. Depletion of SMUG1, and, in particular, the combined loss of SMUG1 and DKC1, leads to accumulation of 5-hydroxymethyluridine in rRNA. In conclusion, SMUG1 is a DKC1 interaction partner that contributes to rRNA quality control, partly by regulating 5-hydroxymethyluridine levels.

Human U1 snRNA forms a new chromatin-associated snRNP with TAF15.

The U1 small nuclear RNA (snRNA)--in the form of the U1 spliceosomal Sm small nuclear ribonucleoprotein particle (snRNP) that contains seven Sm and three U1-specific RNP proteins-has a crucial function in the recognition and removal of pre-messenger RNA introns. Here, we show that a fraction of human U1 snRNA specifically associates with the nuclear RNA-binding protein TBP-associated factor 15 (TAF15). We show that none of the known protein components of the spliceosomal U1-Sm snRNP interacts with the newly identified U1-TAF15 snRNP. In addition, the U1-TAF15 snRNP tightly associates with chromatin in an RNA-dependent manner and accumulates in nucleolar caps upon transcriptional inhibition. The Sm-binding motif of U1 snRNA is essential for the biogenesis of both U1-Sm and U1-TAF15 snRNPs, suggesting that the U1-TAF15 particle is produced by remodelling of the U1-Sm snRNP. A demonstration that human U1 snRNA forms at least two structurally distinct snRNPs supports the idea that the U1 snRNA has many nuclear functions.

PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function.

TAF15 (formerly TAF(II)68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.