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

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 recyclin

Uridylation of miRNAs by HEN1 SUPPRESSOR1 in \u3ci\u3eArabidopsis\u3c/i\u3e

HEN1-mediated 2′-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3′ terminal uridylation [1–8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3′ end of RNA, which is completely abolished by 2′-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3′-to-5′ exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals

Function and Regulation of Human Terminal Uridylyltransferases

RNA uridylylation plays a pivotal role in the biogenesis and metabolism of functional RNAs, and regulates cellular gene expression. RNA uridylylation is catalyzed by a subset of proteins from the non-canonical terminal nucleotidyltransferase family. In human, three proteins (TUT1, TUT4, and TUT7) have been shown to exhibit template-independent uridylylation activity at 3′-end of specific RNAs. TUT1 catalyzes oligo-uridylylation of U6 small nuclear (sn) RNA, which catalyzes mRNA splicing. Oligo-uridylylation of U6 snRNA is required for U6 snRNA maturation, U4/U6-di-snRNP formation, and U6 snRNA recycling during mRNA splicing. TUT4 and TUT7 catalyze mono- or oligo-uridylylation of precursor let-7 (pre–let-7). Let-7 RNA is broadly expressed in somatic cells and regulates cellular proliferation and differentiation. Mono-uridylylation of pre–let-7 by TUT4/7 promotes subsequent Dicer processing to up-regulate let-7 biogenesis. Oligo-uridylylation of pre–let-7 by TUT4/7 is dependent on an RNA-binding protein, Lin28. Oligo-uridylylated pre–let-7 is less responsive to processing by Dicer and degraded by an exonuclease DIS3L2. As a result, let-7 expression is repressed. Uridylylation of pre–let-7 depends on the context of the 3′-region of pre–let-7 and cell type. In this review, we focus on the 3′ uridylylation of U6 snRNA and pre-let-7, and describe the current understanding of mechanism of activity and regulation of human TUT1 and TUT4/7, based on their crystal structures that have been recently solved

3′ RNA Uridylation in Epitranscriptomics, Gene Regulation, and Disease

Emerging evidence implicates a wide range of post-transcriptional RNA modifications that play crucial roles in fundamental biological processes including regulating gene expression. Collectively, they are known as epitranscriptomics. Recent studies implicate 3′ RNA uridylation, the non-templated addition of uridine(s) to the terminal end of RNA, as a key player in epitranscriptomics. In this review, we describe the functional roles and significance of 3′ terminal RNA uridylation that has diverse functions in regulating both mRNAs and non-coding RNAs. In mammals, three Terminal Uridylyl Transferases (TUTases) are primarily responsible for 3′ RNA uridylation. These enzymes are also referred to as polyU polymerases. TUTase 1 (TUT1) is implicated in U6 snRNA maturation via uridylation. The TUTases TUT4 and/or TUT7 are the predominant mediators of all other cellular uridylation. Terminal uridylation promotes turnover for many polyadenylated mRNAs, replication-dependent histone mRNAs that lack polyA-tails, and aberrant structured noncoding RNAs. In addition, uridylation regulates biogenesis of a subset of microRNAs and generates isomiRs, sequent variant microRNAs that have altered function in specific cases. For example, the RNA binding protein and proto-oncogene LIN28A and TUT4 work together to polyuridylate pre-let-7, thereby blocking biogenesis and function of the tumor suppressor let-7 microRNA family. In contrast, monouridylation of Group II pre-miRNAs creates an optimal 3′ overhang that promotes recognition and subsequent cleavage by the Dicer-TRBP complex that then yields the mature microRNA. Also, uridylation may play a role in non-canonical microRNA biogenesis. The overall significance of 3′ RNA uridylation is discussed with an emphasis on mammalian development, gene regulation, and disease, including cancer and Perlman syndrome. We also introduce recent changes to the HUGO-approved gene names for multiple terminal nucleotidyl transferases that affects in part TUTase nomenclature (TUT1/TENT1, TENT2/PAPD4/GLD2, TUT4/ZCCHC11/TENT3A, TUT7/ZCCHC6/TENT3B, TENT4A/PAPD7, TENT4B/PAPD5, TENT5A/FAM46A, TENT5B/FAM46B, TENT5C/FAM46C, TENT5D/FAM46D, MTPAP/TENT6/PAPD1)

The Functional Characterization of the N-terminal Domains of TUT4

Cells have the ability to adapt in response to environmental stressors by regulating RNA stability. Terminal uridylyltransferases (TUTases) have emerged as essential enzymes in post-transcriptional regulation. TUTases catalyze the untemplated addition of uridine residues to the RNA 3’-end, which generally leads to RNA degradation. Human TUTase 4 (TUT4) regulates mRNA and miRNA stability by initiating the decay of RNA through the addition of a poly(U) tail. TUT4 encodes two catalytic regions. Previously, the C-terminal catalytic motif was thought to execute uridylation activity, while the N-terminal motif was thought to be catalytically inactive. I here demonstrate that while less active than its Cterminal counterpart, the N-terminal motif is indeed capable of post-transcriptional RNA editing and displays RNA substrate specificity. I further identified one of the three catalytic aspartates required for uridylation activity. This reveals a previously unknown catalytic function of the N-terminal catalytic domains with implications for its biological function

Roles for Terminal Uridyl Transferases in the Post-Transcriptional Regulation of Developmental miRNAs

MicroRNAs (miRNAs) are a diverse and evolutionarily conserved class of non-coding RNAs that play a multitude of roles in many branches of eukaryotic biology. The regulation of miRNAs is dynamically controlled both spatially and temporally, and the expression of miRNAs can be modulated at the level of transcription or at points downstream of the miRNA maturation process. A relevant example of post-transcriptional miRNA regulation is the blockade of let-7 precursor miRNAs by Lin28 in embryonic stem cells. This pathway, which is initiated by the small RNA-binding protein Lin28, recruits the terminal uridyl transferase (TUTase) Zcchc11 to add a non-templated oligouridine tail to the miRNAs 3' end, and signals it for degradation by the cytoplasmic exonuclease Dis3l2. The Lin28/let-7 axis is essential for development and metabolic homeostasis, and is reactivated in a subset of human cancers. This thesis describes the biochemical mechanism underlying Lin28-mediated degradation of let-7, as well as a novel role for Zcchc11 and the related TUTase Zcchc6 in targeting mature developmental miRNAs in a Lin28-independent manner

The Nefarious Nexus of Noncoding RNAs in Cancer

The past decade has witnessed enormous progress, which has seen the noncoding RNAs (ncRNAs) turn from the so called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer

Regulation of RNA stability by terminal nucleotidyltransferases

The dysregulation of RNAs has global effects on all cellular pathways. The regulation of RNA metabolism is thus tightly controlled. Terminal RNA nucleotidyltransferases (TENTs) regulate RNA stability and activity through the addition of non-templated nucleotides to the 3′-end. TENT-catalyzed adenylation and uridylation have opposing effects; adenylation stabilizes while uridylation silences or degrades RNA. All TENT homologs were initially characterized as adenylyltransferases; the identification of caffeine-induced death suppressor protein 1 (Cid1) in Schizosaccharomyces pombe as an uridylyltransferase led to the reclassification of many TENTs as uridylyltransferases. Cid1 uridylates mRNAs that are subsequently degraded by the exonuclease Dis-like 3′-5′ exonuclease 2 (Dis3L2), while the human homolog germline-development 2 (Gld2) has been associated with adenylation of mRNAs and miRNAs and uridylation of Group II pre-miRNAs. Mechanisms regulating these enzymes and the extent of TENT activity on cellular RNA homeostasis remain largely unknown. In this thesis, the regulation of human Gld2 and the role of the yeast Cid1/Dis3L2-mediated RNA decay pathway were investigated. An enzyme kinetic study revealed that Gld2 is a true adenylyltransferase with only weak activity for UTP. A detailed phylogenetic analysis revealed that uridylyltransferases arose multiple times during evolution through a single histidine insertion in the active site of adenylyltransferases. Insertion of the critical histidine into Gld2 changed its nucleotide preference from ATP to UTP. Next, the regulation of Gld2 through site-specific phosphorylation in the predicted disordered N-terminal domain was investigated using phosphomimetic substitutions at specific serine (S) residues. Two sites (S62, S110) increased Gld2 activity while one site (S116) drastically reduced 3′-adenylation activity. Mass spectrometry and in vitro activity assays identified protein kinases A (PKA) and B (Akt1) as kinases that specifically phosphorylate Gld2 at S116 to obliterate nucleotide addition activity similarly to the S116E phosphomimetic mutant. Finally, RNA deep sequencing of cid1 and dis3L2 S. pombe deletion strains revealed that the role of Cid1 is redundant in uridylation-dependent mRNA decay while Dis3L2 is the bottleneck to RNA decay. Deletion of either gene increases the accumulation of misfolded proteins but only the dis3L2 deletion up-regulates stress response proteins. Overall, this thesis demonstrates how terminal nucleotidyltransferases regulate RNA stability