Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.
Self-cleaving ribozymes are well-conserved and have important biological functions, for example in rolling circle replication of circular RNAs, as part of mobile elements or in gene regulation of mRNA biosynthesis (Weinberg et al. 2019). Although some biological functions have been deciphered, for many self-cleaving ribozyme representatives they remain to be elucidated (Weinberg 2021). Self-cleaving ribozymes cleave their own phosphodiester backbone site-specifically. In this SN2-like reaction, the nucleophilic attack of the 2ʹ oxygen on the adjacent phosphorous center results in two cleavage products: the 5ʹ fragment with a 2ʹ,3ʹ cyclic phosphate (2ʹ,3ʹ cP) and the 3ʹ fragment with a 5ʹ hydroxyl (5ʹ OH) end (Ferré-D’Amaré and Scott 2010). To date, 10 natural self-cleaving ribozyme classes are known, which were mostly found by studying biological systems. Some self-cleaving ribozymes, namely pistol, twister sister and hatchet were found by targeted searches using comparative genomics analysis (Weinberg et al. 2015). In addition to this targeted bioinformatics analysis, genome-wide experimental approaches were developed to investigate self-cleaving ribozymes (Chen et al. 2021; Salehi-Ashtiani et al. 2006). Lately, Kapranov and colleagues described a method where they used fragmented genomic DNA to which they attached known sequences including the T7 promoter (Chen et al. 2021). These DNAs were PCR amplified, in vitro transcribed and the resulting RNA transcripts were enzymatically treated with RNA 5ʹ pyrophosphohydrolase (RppH) and 3ʹ–5ʹ exonuclease (Xrn1) to enrich 5ʹ OH RNAs derived, for example, from self-cleavage. This method led to the discovery of the latest self-cleaving ribozyme class, which was found in a human long non-coding RNA (lncRNA) – the hovlinc ribozyme. Recently, another approach, called cyPhyRNA-seq, was introduced which enables the direct capture of both ribozyme cleavage fragments from total RNA (Olzog et al. 2021). In cyPhyRNA-seq, the 5ʹ fragment with a 2ʹ,3ʹ cP end is specifically targeted by a variant of Arabidopsis thaliana tRNA ligase (AtRNL AA). To capture the 3ʹ fragment, 5ʹ OH RNAs are enriched by RppH and Xrn1 treatment and ‘tRNA blocking’ (Olzog et al. 2021). In ‘tRNA blocking’, a hairpin adapter with a sequence complementary to the CCA end of tRNAs is ligated to mature tRNAs by the T4 DNA ligase (Erber et al. 2020), thus eliminating mature tRNAs from the pool of ligatable molecules. The enriched 5ʹ OH RNAs are then targeted by adapter ligation with T4 RNA ligase 2 truncated KQ (T4 RNL2 TKQ). Contrary to the adapter ligation to the 5ʹ ribozyme fragment, the ligation to the 3ʹ ribozyme fragment takes place at the 3ʹ OH end, which is also found on most cellular RNAs. This potentially results in the accumulation of a high number of reads that are not self-cleaving ribozyme-derived. Although 5ʹ OH RNAs are enriched by degrading 5ʹ tri- (5ʹ PPP) or monophosphate (5ʹ P)-containing RNAs using RppH and Xrn1, there can be exceptions, such as mature tRNAs that have a recessed 5ʹ phosphate. While ‘tRNA blocking’ offers an efficient way of eliminating mature tRNAs from the pool of reads, there are likely other RNAs that remain after RppH and Xrn1 treatment, that do not possess a 5ʹ OH. Therefore, an alternative approach directly ligating the adapter to the RNA’s 5ʹ OH end would be advantageous. An enzyme that could serve this purpose is the RtcB ligase. This ligase joins RNAs with 2ʹ,3ʹ cP to RNAs with 5ʹ OH using GTP and Mn(II) (Tanaka and Shuman 2011). First, the ligase reacts with GTP to form an RtcB-pG intermediate. In the second step, the RtcB-pG hydrolyzes the 2ʹ,3ʹ cP and transfers the GMP to the 3ʹ phosphate (3ʹ P) of the first RNA. Then, the 3ʹ the guanylated RNA (RNAppG) is ligated to the 5ʹ OH end of the second RNA (Englert et al. 2012; Tanaka et al. 2011a, 2011b). RtcB ligase is found in all domains of life, except fungi and most plants (Tanaka et al. 2011a, 2011b). In prokaryotes, it is assumed that the RtcB ligase is involved in the RNA repair pathway in response to cellular stress (Chakravarty et al. 2012; Tanaka and Shuman 2011). For Escherichia coli (E. coli), it is reported that RtcB has a significant role in ribosome homeostasis and in the repair of cleaved rRNAs by the ribotoxin MazF (Engl et al. 2016; Temmel et al. 2017). In metazoa and possibly archaea, RtcB ligase plays an important role in the ligation of spliced tRNAs (Englert et al. 2011; Popow et al. 2011). Furthermore, the ligase is involved in the unfolded protein response initiated by mRNA splicing of X-box binding protein 1 (XBP1) in metazoa (Kosmaczewski et al. 2014; Lu et al. 2014). In some organisms, the gene encoding RtcB ligase is located in one operon with a gene coding for the protein Archease (Desai et al. 2014). In presence of the Archease protein, the RtcB ligase is able to catalyze multiple turnover reactions. However, RtcB in E. coli and some other bacteria is Archease-independent and consequently, can only perform single turnover ligations. Desai and coworkers also demonstrated the need for RtcB and Archease to coevolve for producing a functional interaction (Desai et al. 2015). Therefore, it has been shown that Archease-independent RtcB ligases, such as the one from E. coli, cannot use Archeases from other organisms as cofactor to enhance their own activity.
Here, we explore the use of RtcB ligases for capturing 3ʹ ribozyme fragments in genome-wide ribozyme screenings, such as cyPhyRNA-seq. We compare E. coli RtcB ligase (EcoRtcB) with the Pyrococcus horikoshii RtcB ligase (PhoRtcB) and Archease for their ability to ligate adapters to the 5ʹ OH end of 3ʹ ribozyme fragments. Additionally, we evaluate DNA, RNA or 2ʹ modified RNA for their use as adapter molecules in RtcB ligations. Then, we apply our optimized conditions to capture 3ʹ ribozyme fragments from E. coli total RNA in a transcriptome-wide approach and compare it to the currently used enrichment strategy for the capture of 3ʹ ribozyme fragments in cyPhyRNA-seq. Although we were able to detect spiked ribozyme fragments when applying the RtcB ligation strategy, the cyPhyRNA-seq method was more efficient in comparison. Nevertheless, the use of RtcB ligase represents an alternative approach for the analysis of other (highly structured) RNAs with 5ʹ OH termini. Especially, PhoRtcB with Archease could provide a promising ligase for the targeted investigation of non-ribozyme derived 5ʹ OH-containing structured RNAs.
Results and discussion
Adapters are efficiently ligated to structured ribozyme fragments by the thermostable RtcB ligase and Archease from P. horikoshii
Previously, EcoRtcB has been used for a high-throughput sequencing approach, where a DNA adapter with a sequence of eight randomized RNA nucleotides at its 3ʹ end is ligated to RNAs with 5ʹ OH termini (Peach et al. 2015). With this method, novel 5ʹ OH fragments derived from mRNAs were identified. However, in contrast to the adapter ligation to mRNAs, ligation to highly structured ribozyme fragments appears more challenging. Therefore, we investigated and optimized the ligation of an RNA adapter to 3ʹ ribozyme cleavage fragments with diverse structures using EcoRtcB and PhoRtcB with Archease (Figure 1). In addition to previously published reaction conditions, we added 15% PEG8000.
Our results demonstrate that EcoRtcB was able to join the RNA adapter to the 3ʹ twister type P5 or 3ʹ hatchet ribozyme fragment, but nearly no ligation products of the 3ʹ twister type P1 and 3ʹ HDV ribozyme fragments were detectable. One reason for the failed ligation could be the complex structures of these ribozymes even after cleavage. For example, the 3ʹ cleavage product of the twister ribozyme type P1 can still form both pseudoknots (Roth et al. 2014) and the 3ʹ HDV ribozyme fragment that comprises most of the ribozyme can still fold into its complex structure after cleavage.
In contrast to the EcoRtcB, the PhoRtcB ligase was able to ligate the RNA adapter to all of the used 3ʹ ribozyme fragments. Firstly, the more efficient ligation of the PhoRtcB ligase is possibly achieved by its conversion into an enzyme catalyzing multiple turnovers in presence of the cofactor Archease, which increases the ligation activity (Desai et al. 2014). Because EcoRtcB ligation is a single-turnover reaction, a higher concentration of this enzyme would have to be used to get a comparable ligation efficiency. Secondly, PhoRtcB is thermostable and therefore active at temperatures well above 37 °C. Due to the higher incubation temperature (usually 70 °C) possible with PhoRtcB, RNA’s secondary and tertiary structures unfold and thus, the relevant RNA ends are more accessible for ligation. Taken together, using a thermostable RtcB ligase together with Archease enables efficient ligation of adapters to structured RNAs such as ribozyme fragments.
RtcB ligases do not efficiently ligate DNA adapters to single-stranded DNA or RNA molecules
Some self-cleaving ribozymes like HDV and hatchet cleave themselves near the 5ʹ end (Ferré-D’Amaré et al. 1998; Zheng et al. 2019). Because these ribozymes can still fold into their active structure after cleavage, they could be capable of cutting off RNA adapters immediately or in subsequent reactions. To prove this, we PAGE-purified RtcB-mediated adapter-hatchet ligation product and incubated it for 2 h in Mg2+-containing buffer used for the second adapter ligation. In this experiment, we observed – apart from the ligation product – the reappearance of hatchet ribozyme fragment that was likely liberated from the RNA adapter by self-cleavage (Supplementary Figure S1). As the removal of an adapter from the ribozyme fragment is problematic for ribozyme analysis by RNA-seq, we subsequently investigated the ligation of DNA and different 2ʹ modified RNA adapters to 3ʹ ribozyme fragments.
When applying a DNA instead of an RNA adapter, the catalysis of self-cleavage is impossible due to the lack of the 2ʹ O nucleophile. Therefore, we investigated the ability of ligating DNA adapters that are 5ʹ-labeled with [γ32P]-ATP to RNA and DNA substrates by EcoRtcB ligase as well as PhoRtcB (Figure 2). Wherever PhoRtcB is indicated, the reactions included the RtcB enzyme and its Archease. For the initial analysis, we used a simplified model adapter/substrate pair capable of base-pairing similarly to previously used ligation substrates (Tanaka and Shuman 2011). These nucleic acid molecules closely mimic the natural RtcB substrate of a tRNA anticodon stem-loop and should enable optimal ligation. In the past, ligation experiments with EcoRtcB demonstrated that only at least partly complementary DNAs were ligated resulting in a hairpin (Das et al. 2013). Our experiments confirm that EcoRtcB was able to ligate the DNA adapter to DNA or RNA substrate only when both strands are part of a common stem-loop structure. However, this ligation is not as efficient as if joining two RNAs. In contrast to EcoRtcB, the analogous ligation of a DNA adapter to DNA or RNA substrate by PhoRtcB was not successful. Attempts at joining a DNA adapter with a 3ʹ ribozyme fragment using EcoRtcB failed (data not shown).
Unlike the ligation reaction, the guanylation of the DNA adapter was very efficient for both ligases (Figure 2B and C). This stable DNA guanylation was already described in earlier publications and is due to the 2ʹ H (Das and Shuman 2013). In contrast, for guanylated RNA it is assumed that the released RNAppG would rapidly form 2ʹ,3ʹ cP in solution via a nucleophilic attack by the terminal 2ʹ OH (Desai et al. 2014). Interestingly, it seems that even a second GMP was transferred to the DNA adapter by EcoRtcB because a shift by one additional nucleotide was detectable in the gel (Figure 2B). For PhoRtcB, only a faint band is visible, likely corresponding to GppDNAppG. Therefore, we analyzed if GMP can be transferred to the 5ʹ P of DNAs (Figure 2C). In this experiment, we used a 5ʹ labeled DNA adapter with a 3ʹ amino protection group and observed that GMP was transferred to the 5ʹ P. Nevertheless, this 5ʹ guanylated DNA adapter was not joined to a DNA substrate with 5ʹ or 3ʹ OH end. Hence, EcoRtcB cannot only transfer GMP to the 3ʹ P, but also to the 5ʹ P of DNA adapters. Previously, an ability of RtcB ligase to transfer GMP to the 5ʹ P and 3ʹ P of a DNA strand has been observed only for an RtcB paralog (RtcB3) from Myxococcus xanthus (Maughan and Shuman 2015). For EcoRtcB a 5ʹ P guanylation was not detected in previous experiments, which used a 5ʹ P DNA oligonucleotide that perfectly annealed at its 5ʹ end with the second DNA strand (Das et al. 2013). We assume that under these conditions the 5ʹ P might not be accessible for guanylation by RtcB and thus, that at least one non-complementary base might be necessary for EcoRtcB to enable the GMP-transfer to a 5ʹ P DNA end.
The ability of a ligase to activate an RNA at either end (the 5ʹ P and the 3ʹ P) is not entirely unprecedented. It was already observed for other enzymes like RtcA cyclase and T4 RNL. In E. coli, RtcA is encoded in one operon with RtcB and it usually converts 3ʹ P ends to 2ʹ,3ʹ cP by adenylating the 3ʹ P (Genschik et al. 1998). However, RtcA has also been shown to adenylate 5ʹ P ends of DNA as well as RNA substrates (Chakravarty and Shuman 2011). The T4 RNL normally adenylates 5ʹ P ends to form 3ʹ–5ʹ phosphodiester bonds between a nucleic acid with a 3ʹ OH and another with a 5ʹ P. This enzyme was also found to produce unusual 3ʹ adenylated products (Hinton et al. 1982).
RtcB ligases do not efficiently ligate 2ʹ modified RNA adapters to ribozyme fragments
One explanation for the low ligation efficiency of DNA to RNA by RtcB could lie in an inability of the enzyme to accommodate binding of a single-stranded DNA. Accordingly, we investigated the ability of RtcB enzymes to ligate RNA adapters that merely contained different 2ʹ modifications instead of the 2ʹ OH at their 3ʹ terminal nucleotide (Supplementary Figure S2). Because the 2ʹ end is modified, no nucleophilic attack can occur and thus even ribozymes such as hatchet or HDV would not be able to support the liberation of the ligated adapter by cleavage.
We used an RNA adapter with either (i) a 3ʹ terminal DNA nucleotide (2ʹ H), (ii) a 2ʹ O-methyl group (2ʹ OMe), (iii) a 2ʹ fluoro group or (iv) a 3ʹ terminal locked nucleic acid (LNA) nucleotide (Supplementary Figure S2). We employed the 3ʹ fragment of a hatchet and twister type P5 ribozyme, which were previously proven to be suitable ligation substrates (Figure 1). High amounts of guanylated RNA adapter with 2ʹ H (PhoRtcB) and 2ʹ F (both ligases) were detectable, especially in comparison to the unmodified RNA adapter (Supplementary Figure S2). In contrast to PhoRtcB, EcoRtcB is able to guanylate all 2ʹ modified adapters.
Moreover, we found that none of the 2ʹ modified RNA adapters were efficiently ligated to ribozyme fragments. Therefore, the presence of a 2ʹ OH group appears to be crucial for ligation. However, the specific role the 2ʹ OH could play in the ligation mechanism is currently unclear. The most likely ligation mechanism relies on the opening of the 2ʹ,3ʹ cP followed by the activation of the resulting 3ʹ P by guanylation (Chakravarty et al. 2012; Tanaka et al. 2011a, 2011b). Then the 5ʹ OH drives a nucleophilic attack resulting in the connection of the two RNA ends in a 3ʹ–5ʹ phosphodiester bond. To our knowledge the only RtcB-related reaction described that involves the 2ʹ OH is the reversal of the cyclic phosphodiesterase reaction observed when RNA ligation is prevented by a 5ʹ P (Tanaka et al. 2011a, 2011b). In this case, the 2ʹ OH acts as a nucleophile for the attack on the 3ʹ guanylated RNA and a 2ʹ,3ʹ cP is formed again. An alternative ligation mechanism having this reaction at its core (Desai and Raines 2012) was soon shown to be invalid (Chakravarty and Shuman 2012). The more efficient RNA ligation underscores that the 2ʹ OH increases the ligation rate directly or indirectly and an influence on the conformation of the terminal pentose sugar has been suggested (Chakravarty et al. 2012). This however leaves to be determined why an RNA with a terminal LNA nucleotide also does not support ligation, as in this case the pentose is locked in a C3ʹ endo conformation analogous to ribose in RNA (Supplementary Figure S2). Possibly a loss of hydrogen-bonding between RtcB and the 2ʹ OH could cause the decline in ligation efficiency. This increases the need for solving a crystal structure of RtcB that gives insight into substrate binding at the 3ʹ end and the exact role of the 2ʹ OH. Additionally, such information might help to predict an altered 2′ group that permits ligation.
The 3ʹ twister ribozyme fragment was captured from E. coli total RNA by RtcB-mediated ligation
Since we showed that only RNA adapters are suitable for the ligation to the 5ʹ OH end of ribozymes, we used this adapter in further experiments. We then applied the RtcB ligation to capture a spiked 3ʹ twister type P5 ribozyme fragment from E. coli total RNA and compared it to the previously described cyPhyRNA-seq method. cyPhyRNA-seq includes two parallel strategies, each designed to select either the 5ʹ or 3ʹ ribozyme fragment. To find the 3ʹ fragment, an alternative method is applied in which the adapter is not ligated directly to the 5ʹ OH end. Instead, 5ʹ OH-RNAs in total RNA are first enriched by degrading RNAs with 5ʹ tri- and monophosphates using RppH and Xrn1. Then, the 5ʹ adenylated adapter is ligated to the 3ʹ OH end of the enriched 5ʹ OH containing RNAs by T4 RNL2 TKQ (Olzog et al. 2021). In contrast, RtcB ligation can serve as an alternative approach, in which the adapter ligation occurs directly at the 5ʹ OH end.
In the RtcB ligation method, E. coli total RNA spiked with the 3ʹ twister type P5 ribozyme fragment was enriched for short RNAs by precipitating large RNAs with PEG8000 and NaCl (Lu et al. 2007). The samples were then used either directly for adapter ligation by RtcB or for ’tRNA blocking’ (Olzog et al. 2021). Using these approaches, large rRNAs and mature tRNAs, which are the most abundant RNA types in total RNA samples (O’Neil et al. 2013; Westermann et al. 2012), can be efficiently removed. The enriched RNA is then used for producing high-throughput sequencing libraries (Figure 3A). First, the RNA adapter is ligated to the 3ʹ ribozyme fragment by PhoRtcB ligase. Next, the second, DNA adapter with a unique molecular identifier (UMI) (Kivioja et al. 2011), which is pre-adenylated by TS2126 RNA ligase 1, is ligated to the 3ʹ OH of the ribozyme fragment with T4 RNL2 TKQ. After adapter ligation to both ends of the ribozyme fragment, the ligation product is reverse transcribed by SuperScript IV (SSIV) reverse transcriptase, subsequently PAGE-purified and used as template for PCR amplification by Phusion DNA Polymerase (Figure 3A). The produced libraries were used for Amplicon-EZ sequencing with ∼50,000 to 100,000 reads (Azenta Life Sciences). After adapter trimming and mapping, the reads were deduplicated, which means that only reads with different UMIs and thus, single ligation events, were considered. Furthermore, we only investigated uniquely mapping reads. Based on the annotation, the deduplicated and uniquely mapping reads were assigned to general RNA classes: rRNAs, tRNAs, mRNAs, other non-coding RNAs and the spiked twister type P5 ribozyme fragments (Figure 3B, Supplementary Table S1).
In all prepared libraries, we were able to detect the spiked 3ʹ twister type P5 ribozyme fragment. The ‘tRNA blocking’ results in a significant reduction of tRNA reads, which we also detected in our RtcB-based method (Figure 3B). When tRNAs are not blocked, 55% of the reads belong to tRNAs, while only 8% of all reads correspond to tRNAs when tRNA blocking was performed. Looking at the percentage of RNA types found, it appears that more rRNAs are observed when ‘tRNA blocking’ is performed. However, the actual number of reads changes only slightly, while the total number of unique and deduplicated reads decreases because fewer tRNA-based reads are generated (Supplementary Table S1). This means, using ‘tRNA blocking’, rarer RNAs can potentially be uncovered as fewer reads are siphoned off by tRNAs. Compared to the RNA enrichment followed by T4 RNL2 TKQ-mediated ligation as performed in the cyPhyRNA-seq method, more rRNA and mRNA reads were detected in the RtcB-mediated ligation (Supplementary Table 1). In order to reduce these mRNA and rRNA reads, the enzymatic enrichment of 5ʹ OH RNAs by RppH and Xrn1 in contrast to the currently used precipitation of large RNAs could be investigated in future. Furthermore, the higher number of detected rRNA and mRNA reads could also be due to increased RNA background degradation during the RtcB ligation reaction that is carried out at 70 °C in 0.5 mM MnCl2. Therefore, further conditions could be adjusted for the use of PhoRtcB in RNA-seq-based methods, e.g. incubation time, reaction buffer (MOPS instead of Tris as published in Peach et al. 2015) or concentration of MnCl2. Furthermore, ‘tRNA blocking’ does not lead to an increased number of captured ribozyme fragments, because the total number of captured 3ʹ twister type P5 ribozyme fragments is the same in both RtcB-based libraries (Supplementary Table 1). Using the cyPhyRNA-seq method with the T4 RNL2 TKQ, a higher number of 3ʹ twister ribozyme fragments was observed (16%) compared to the RtcB-based strategy (2%) (Figure 3B).
Use of RtcB-mediated ligations for self-cleaving ribozyme discovery and validation
Previously published methods for capturing ribozyme fragments with 5ʹ OH ends are based on RNA enrichment by enzymatic degradation of ribonucleic acids with 5ʹ PPP and 5ʹ P (Chen et al. 2021; Olzog et al. 2021). In contrast, RtcB ligase can be used to directly ligate adapters to the RNA’s 5ʹ OH end. By using EcoRtcB for RNA sequencing, 5ʹ OH-containing tRNA and mRNA fragments have been detected previously (Peach et al. 2015). Here, we apply the RtcB-mediated ligation to capture ribozyme cleavage fragments. We determined that the adapter ligation to highly structured RNAs, such as ribozyme fragments, is much more efficient with the thermostable PhoRtcB together with its Archease enzyme. The Archease is a cofactor of the PhoRtcB that facilitates a multiple turnover reaction and thus, enhances the ligation efficiency (Desai et al. 2014). However, we were not able to use DNA or 2ʹ modified RNA adapters for the ligation to ribozyme fragments. Some self-cleaving ribozyme classes retain their cleavage ability after self-scission and thus can liberate the RtcB-ligated RNA adapters. This deficit renders the RtcB-dependent ligation method less favorable for the application to self-cleaving ribozyme validation and discovery as some classes would escape detection. Nevertheless, using the PhoRtcB-based ligation, we captured spiked ribozyme fragments from total RNA. However, we demonstrate that the latest cyPhyRNA-seq method utilizing enzymatic enrichment is more efficient in capturing ribozyme fragments with 5ʹ OH, even though it does not rely on a direct ligation to RNAs with 5ʹ OH termini.
Use of RtcB-mediated ligations for screening 5ʹ OH RNAs
Even if the use of RtcB is not ideal for ribozymes, the use of PhoRtcB for the ligation-directed capture of 5ʹ OH RNAs could be useful for studying other RNAs. Identical termini are produced in a number of cellular processes (Shigematsu et al. 2018) or treatments (Lindell et al. 2002; Twittenhoff et al. 2020), for example by certain endoribonucleases (e.g. RNase A or T2) (Luhtala and Parker 2010), in stress-induced cleavage of tRNAs (e.g. oxidative stress) (Thompson et al. 2008) and in spontaneous degradation. Additionally, it could be a great complementary approach for further applications where RNAs with 2ʹ,3ʹ cP are captured by AtRNL AA (Olzog et al. 2021). Although, EcoRtcB has been previously used for transcriptome-wide detection of RNAs with 5ʹ OH (Peach et al. 2015), the use of PhoRtcB offers three advantages. First, elevated incubation temperatures enable ligation of even highly structured RNAs, second, the Archease cofactor enables PhoRtcB to perform a multiple turnover reaction increasing ligation efficiency and third, both of these factors lead to drastically shortened incubation times (20 min vs. 2 h). Therefore, the PhoRtcB ligation strategy could be used to detect highly structured 5ʹ OH RNAs in vivo on a genome-wide scale.
Taken together, in addition to the existing ribozyme capture strategies (Chen et al. 2021; Olzog et al. 2021), the RtcB-based method has potential for the investigation and detection of self-cleaving ribozymes. Although a few self-cleaving ribozyme classes such as hatchet and HDV cannot be efficiently targeted by RtcB-mediated ligation, more than half of all known natural self-cleaving ribozyme classes do not cleave near their 5ʹ end. Thus, these ribozymes should be detectable by an RNA-seq-based screening strategy that relies on ligation by RtcB. Nevertheless, the RtcB-based method appears even more suitable for the investigation of other RNA species with 5ʹ OH ends.
While investigating RtcB ligases for the application in RNA-seq, we noticed the ability of EcoRtcB to guanylate 5ʹ P ends. This has not been observed previously for this enzyme and in fact has only been studied for an RtcB paralog from Myxocuccus xanthus (Maughan and Shuman 2015). The fact that the E. coli enzyme is also capable of transferring a GMP to RNA 5ʹ ends might suggest that this activity is more universal among bacterial RtcB enzymes than previously assumed.
Lastly, the inability of EcoRtcB to ligate RNA adapters with different 2ʹ modifications to 5ʹ OH RNAs suggests that there is a role for the 2ʹ OH group in enhancing RtcB ligation. It remains to be elucidated how the 2ʹ OH is involved in supporting the ligation by RtcB ligases. Solving a crystal structure of RtcB bound to an RNA where the RNA’s 3ʹ end is resolved could provide clues as to the role of the terminal 2ʹ OH. Additionally, with this knowledge, it may be possible to engineer an RtcB ligase capable of catalyzing the connection of a DNAp with an RNA with 5ʹ OH, making this enzyme not only applicable for the generation of RNA-seq libraries screening for any 5ʹ OH RNAs but also including catalytic RNAs such as self-cleaving ribozymes.
Materials and methods
Expression and purification of E. coli RtcB ligase
The expression and purification of EcoRtcB was performed as described previously (Olzog et al. 2021).
Expression and purification of P. horikoshii RtcB ligase and Archease
The plasmid pET32 with the sequence of P. horikoshii Archease or RtcB ligase (gift from Ronald Raines) was used for transformation of E. coli BL21 (DE3) cells. The overexpressions of PhoRtcB ligase and Archease were carried out in the same way: 4 ml of overnight culture was used to inoculate 800 ml TB medium containing 100 μg/ml ampicillin. The main culture was incubated at 37 °C and 180 rpm until an OD600 of 0.6 was reached. Then, 0.5 mM isopropyl-β-d-thiogalactoside (IPTG) was added and the incubation was continued for 3 h. Cells were harvested by centrifugation for 15 min at 4 °C and 4000g and stored at −80 °C.
Pyrococcus horikoshii RtcB ligase was purified as previously described (Desai and Raines 2012). Pellet was resuspended in Buffer A [50 mM Tris-HCl (pH 7.7), 300 mM NaCl, 0.5 mM DTT and 25 mM imidazole] (6 ml per g of wet pellet). Cells were disrupted by a Fastprep-24 Homogenisator (MP Biomedicals GmbH, Eschwege, Germany) for 6 m/s and 30 s. After centrifugation for 30 min at 30,600g and 4 °C, supernatant was sterile filtered and loaded onto HisTrap FastFlow column (1 ml, GE Healthcare, Freiburg, Germany) equilibrated with Buffer A. The column was washed with 40 mM imidazole (15% Buffer B). Elution was performed with Buffer B [50 mM Tris-HCl (pH 7.7), 300 mM NaCl, 0.5 mM DTT and 250 mM imidazole]. Peak fractions were pooled and used for size exclusion chromatography (SEC) using HiLoad 16/60 Superdex 75 pg (GE Healthcare) column equilibrated in Buffer C [20 mM Tris-HCl (pH 7.4), 300 mM NaCl]. Peak fractions were combined and concentrated using Vivaspin 6 (10 kDa cutoff). Aliquots of 50 µl were frozen in liquid nitrogen and stored at −80 °C.
Pyrococcus horikoshii Archease was purified as previously described (Desai et al. 2015). Pellet was resuspended in Buffer D [50 mM Tris-HCl (pH 7.7), 300 mM NaCl and 20 mM imidazole] (6 ml per g of wet pellet) and cells were disrupted by a Fastprep-24 Homogenisator for 6 m/s and 30 s. After centrifugation for 30 min at 30,600g and 4 °C, supernatant was used for a heat-kill step at 70 °C for 25 min. After another centrifugation at 30,600g and 4 °C for 20 min, supernatant was sterile filtered and loaded onto HisTrap FastFlow column (5 ml, GE Healthcare) equilibrated with Buffer D. The column was washed with 45 mM imidazole (18% Buffer E). Elution was performed with Buffer E [50 mM Tris-HCl (pH 7.7), 300 mM NaCl and 250 mM imidazole]. Peak fractions were pooled and dialyzed in Buffer F [10 mM HEPES-NaOH (pH 7.5) and 200 mM NaCl] overnight at 4 °C. Aliquots of 50 µl were frozen in liquid nitrogen and stored at −80 °C.
RNA synthesis and preparation of oligonucleotides
Oligonucleotides were ordered from biomers.net (Ulm, Germany) or Microsynth AG (Balgach, Switzerland). Templates for ribozymes were generated by PCR amplification using a 50 µl PCR reaction containing 1× Pfu DNA Polymerase Buffer [20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1 mg/ml BSA, 2 mM MgSO4 × 7 H2O], 200 µM dNTPs, 4 or 0.4 µM forward and reverse primer (Supplementary Table S2) and 1 µl of the Pfu DNA Polymerase (own laboratory purification). Initial denaturation step was performed at 94 °C for 5 min. Subsequent PCR cycles include a denaturation step at 94 °C for 10 s, annealing step at the respective annealing temperature for 10 s, elongation step at 68 °C for 15–30 s and were performed for 25 cycles. Final elongation step was carried out at 68 °C for 2 min. PCR reactions were purified using phenol/chloroform extraction and ethanol precipitation (Sambrook and Russell 2006).
Adapters were radioactively labeled using [γ32P]-ATP (Hartmann Analytic) and T4 Polynucleotide Kinase D167N (Das and Shuman 2013). Model substrates and ribozyme cleavage fragments (Supplementary Table S3) were generated by T7-based in vitro transcription using purified PCR products (Ling et al. 1989) and [α32P]-ATP (Hartmann Analytic, Braunschweig, Germany). Then, ribozyme fragments or oligonucleotides were PAGE-purified as described previously (Lihanova and Weinberg 2021).
Ligations using EcoRtcB
First, 1 µM adapter with 3ʹ P, 0.5 µM 3ʹ ribozyme fragment with 5ʹ OH (Supplementary Table S3) and diethylpyrocarbonate (DEPC)-H2O were incubated at 65 °C for 5 min and cooled down on ice. Then, 50 mM Tris-HCl (pH 7.4), 2 mM MnCl2, 100 µM GTP, 15% polyethylene glycol 8000 (PEG8000), 5% dimethyl sulfoxide (DMSO) and 5 µM EcoRtcB were added. Ligation reactions were incubated at 37 °C for 2 h. Reactions were stopped by adding 3× RNA loading dye [10 mM Tris-HCl (pH 7.6 at 23 °C), 80% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol]. The ligation was analyzed by polyacrylamide (PAA) gel electrophoresis using a 15–20% denaturing PAA gel. Ligation efficiency was calculated from measuring band intensities using ImageQuant.
Ligations using PhoRtcB
Adapter with 3ʹ P (1 µM), 3ʹ ribozyme fragment with 5ʹ OH (0.5 µM, Supplementary Table S3) and DEPC-H2O were incubated 5 min at 65 °C and cooled down on ice. Afterwards, 50 mM Tris-HCl (pH 7.4), 0.5 mM MnCl2, 100 µM GTP, 300 mM NaCl, 15% PEG8000, 2 µM Archease and 5 µM RtcB ligase were added to the adapter/substrate-mix. After the incubation at 70 °C for 20 min, ligation reactions were stopped by adding 3× RNA loading dye . The ligation was analyzed by polyacrylamide (PAA) gel electrophoresis using a 15–20% denaturing PAA gel. The ligation efficiency was calculated from measuring band intensities using ImageQuant.
Preparation of E. coli total RNA and twister P5 ribozyme fragment
Escherichia coli TOP10 cells were grown to an OD600 of 0.5 and centrifuged at 4000 g and 4 °C for 10 min. Total RNA was isolated using TRIzol™ reagent (Thermo Scientific, Darmstadt, Germany) according to manufacturer’s instructions. 3ʹ fragment of twister type P5 with 5′-OH (Supplementary Table S3) was produced by in vitro transcription and self-cleavage followed by PAGE-purification and ethanol precipitation. Finally, 1 ng of ribozyme cleavage fragment was spiked into 1 μg E. coli total RNA.
Enrichment of short RNAs from total RNA and ‘tRNA blocking’
Spiked RNA was used for short RNA enrichment using PEG8000 and NaCl. For that, 50% PEG8000 and 5 M NaCl was added to a 100 µl reaction containing 1 μg E. coli total RNA spiked with 1 ng 3ʹ-twister type P5 ribozyme fragment to final concentrations of 5% and 0.5 M (Lu et al. 2007). Samples were incubated at −20 °C for 30 min and subsequently centrifuged 30 min at 10,000g and 4 °C. Afterwards, three volumes of absolute ethanol were added to supernatant and incubated overnight at −20 °C. Then, samples were centrifuged at 17,000g for at least 1 h. Supernatant was removed and pellet was washed with 70% ethanol and centrifuged again for 5 min. Pellet was air-dried and dissolved in 5 µl DEPC-H2O.
For ‘tRNA blocking’, a hairpin adapter (Supplementary Table S2) was ligated to mature tRNAs with CCA-ends using T4 DNA Ligase (NEB, Frankfurt/Main, Germany) (Erber et al. 2020). Samples were purified using phenol/chloroform extraction and ethanol precipitation and dissolved in 5 µl DEPC-H2O (Sambrook and Russell 2006).
For the first adapter ligation, 40 pmol 5ʹ RNA adapter with 3ʹ P (Supplementary Table S2) was added to enriched RNA. The ligation using PhoRtcB was performed as described above. The ligation reaction was purified using the Monarch RNA Cleanup Kit (10 µg, NEB). Afterwards, the 3ʹ DNA adapter with UMI (Supplementary Table S2) was ligated using T4 RNL2 TKQ and ligation reactions were used for reverse transcription with 5ʹ labeled RT primer and SuperScript IV as described previously (Olzog et al. 2021). After purification using 15% PAA gel, samples were used as template for PCR amplification with Phusion DNA polymerase (Thermo Scientific) and primer with partial Illumina adapter sequences (Supplementary Table S2). Libraries were purified using solid phase reverse immobilization beads (Vazyme VAHTS DNA Clean Beads, Nanjing, China) and adjusted to a final concentration of 20 ng/μl each and sequenced with ∼50 000 reads (Amplicon-EZ sequencing, Azenta Life Sciences, Leipzig, Germany).
The method to capture 3ʹ-ribozyme cleavage fragments with PhoRtcB was compared to the previously described cyPhyRNA-seq method, in which RNAs with 5ʹ OH were first enriched and then, used for adapter ligation with T4 RNL2 TKQ (Olzog et al. 2021). Libraries were produced with and without performing ‘tRNA blocking’.
Analysis of reads generated by amplicon sequencing
Libraries of spike-in experiments were analyzed based on Amplicon-EZ sequencing (Amplicon-EZ, Azenta Life Sciences, Supplementary Table S2). The reads were treated as follows: (1) reads were preprocessed with UMItools (Smith et al. 2017) to identify and trim UMI sequences, (2) trimming of remaining adapters with TrimGalore (Felix Krueger), (3) mapping of reads and (4) counting. To map the reads, an artificial genome was created by adding the spiked-in 3ʹ twister type P5 ribozyme fragment sequence (Supplementary Table S3) to the E. coli genome retrieved from https://www.ncbi.nlm.nih.gov/genome/167?genome_assembly_id=753562. Then, the artificial genome was indexed and reads were mapped with STAR (Dobin et al. 2013). Command-line calls for these workflow steps are listed in the Supplementary text. Reads mapping only at one position in the genome were extracted and counted with featureCounts (Liao et al. 2014). A framework for the semi-automated analysis of the data presented herein is available at https://github.com/jfallmann/MONSDA (manuscript in preparation). Last, reads were manually inspected and deduplicated for reads mapping to spiked twister ribozyme fragment.
Raw data of Amplicon-EZ sequencing from E. coli spiked with the 3ʹ twister type P5 ribozyme fragment is uploaded to NCBI:
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: LU1889/4-1
Funding source: Peter und Traudl Engelhorn Stiftung
We would like to thank Ronald Raines for the generous gift of expression plasmids encoding thermophile RtcB and Archease enzymes and Mario Mörl for hosting the growing Weinberg Lab.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Research in the Weinberg Lab is supported by the Peter and Traudl Engelhorn Foundation (fellowship for C.E.W.) and the German Research Foundation (LU1889/4-1 to C.E.W.).
Conflict of interest statement: The authors declare that they have no competing interests.
Chakravarty, A.K. and Shuman, S. (2011). RNA 3-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5-monophosphate ends. J. Biol. Chem. 286: 4117–4122, https://doi.org/10.1074/jbc.m110.196766.Search in Google Scholar PubMed PubMed Central
Chakravarty, A.K. and Shuman, S. (2012). The sequential 2,3-cyclic phosphodiesterase and 3-phosphate/5-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent. Nucleic Acids Res. 40: 8558–8567, https://doi.org/10.1093/nar/gks558.Search in Google Scholar PubMed PubMed Central
Chakravarty, A.K., Subbotin, R., Chait, B.T., and Shuman, S. (2012). RNA ligase RtcB splices 3-phosphate and 5-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3)pp(5)G intermediates. Proc. Natl. Acad. Sci. U.S.A. 109: 6072–6077, https://doi.org/10.1073/pnas.1201207109.Search in Google Scholar PubMed PubMed Central
Chen, Y., Qi, F., Gao, F., Cao, H., Xu, D., Salehi-Ashtiani, K., and Kapranov, P. (2021). Hovlinc is a recently evolved class of ribozyme found in human lncRNA. Nat. Chem. Biol. 17: 601–607, doi:https://doi.org/10.1038/s41589-021-00763-0.Search in Google Scholar PubMed
Das, U., Chakravarty, A.K., Remus, B.S., and Shuman, S. (2013). Rewriting the rules for end joining via enzymatic splicing of DNA 3-PO4 and 5-OH ends. Proc. Natl. Acad. Sci. U.S.A. 110: 20437–20442, https://doi.org/10.1073/pnas.1314289110.Search in Google Scholar PubMed PubMed Central
Das, U. and Shuman, S. (2013). Mechanism of RNA 2,3-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase. Nucleic Acids Res. 41: 355–365, https://doi.org/10.1093/nar/gks977.Search in Google Scholar PubMed PubMed Central
Desai, K.K., Beltrame, A.L., and Raines, R.T. (2015). Coevolution of RtcB and Archease created a multiple-turnover RNA ligase. RNA 21: 1866–1872, https://doi.org/10.1261/rna.052639.115.Search in Google Scholar PubMed PubMed Central
Desai, K.K., Cheng, C.L., Bingman, C.A., Phillips, G.N., and Raines, R.T. (2014). A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation. Nucleic Acids Res. 42: 3931–3942, https://doi.org/10.1093/nar/gkt1375.Search in Google Scholar PubMed PubMed Central
Desai, K.K. and Raines, R.T. (2012). tRNA ligase catalyzes the GTP-dependent ligation of RNA with 3-phosphate and 5-hydroxyl termini. Biochemistry 51: 1333–1335, https://doi.org/10.1021/bi201921a.Search in Google Scholar PubMed PubMed Central
Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15–21, https://doi.org/10.1093/bioinformatics/bts635.Search in Google Scholar PubMed PubMed Central
Engl, C., Schaefer, J., Kotta-Loizou, I., and Buck, M. (2016). Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli. Nucleic Acids Res. 44: 9933–9941, https://doi.org/10.1093/nar/gkw628.Search in Google Scholar PubMed PubMed Central
Englert, M., Sheppard, K., Aslanian, A., Yates, J.R., and Söll, D. (2011). Archaeal 3-phosphate RNA splicing ligase characterization identifies the missing component in tRNA maturation. Proc. Natl. Acad. Sci. U.S.A. 108: 1290–1295, https://doi.org/10.1073/pnas.1018307108.Search in Google Scholar PubMed PubMed Central
Englert, M., Xia, S., Okada, C., Nakamura, A., Tanavde, V., Yao, M., Eom, S.H., Konigsberg, W.H., Söll, D., and Wang, J. (2012). Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3-terminal phosphate and 5-OH. Proc. Natl. Acad. Sci. U.S.A. 109: 15235–15240, https://doi.org/10.1073/pnas.1213795109.Search in Google Scholar PubMed PubMed Central
Erber, L., Hoffmann, A., Fallmann, J., Betat, H., Stadler, P.F., and Mörl, M. (2020). LOTTE-seq (long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3-CCA end for high-throughput sequencing. RNA Biol. 17: 23–32, https://doi.org/10.1080/15476286.2019.1664250.Search in Google Scholar PubMed PubMed Central
Ferré-DAmaré, A.R. and Scott, W.G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor Perspect. Biol. 2: a003574.10.1101/cshperspect.a003574Search in Google Scholar PubMed PubMed Central
Ferré-DAmaré, A.R., Zhou, K., and Doudna, J.A. (1998). Crystal structure of a hepatitis delta virus ribozyme. Nature 395: 567–574.10.1038/26912Search in Google Scholar PubMed
Genschik, P., Drabikowski, K., and Filipowicz, W. (1998). Characterization of the Escherichia coli RNA 3 -terminal phosphate cyclase and its sigma54-regulated operon. J. Biol. Chem. 273: 25516–25526, https://doi.org/10.1074/jbc.273.39.25516.Search in Google Scholar PubMed
Hinton, D.M., Brennan, C.A., and Gumport, R.I. (1982). The preparative synthesis of oligodeoxyribonucleotides using RNA ligase. Nucleic Acids Res. 10: 1877–1894, https://doi.org/10.1093/nar/10.6.1877.Search in Google Scholar PubMed PubMed Central
Felix Krueger. TrimGalore. https://github.com/FelixKrueger/TrimGalore (accessed 09 September 2020).Search in Google Scholar
Kivioja, T., Vähärautio, A., Karlsson, K., Bonke, M., Enge, M., Linnarsson, S., and Taipale, J. (2011). Counting absolute numbers of molecules using unique molecular identifiers. Nat. Methods 9: 72–74, https://doi.org/10.1038/nmeth.1778.Search in Google Scholar PubMed
Kosmaczewski, S.G., Edwards, T.J., Han, S.M., Eckwahl, M.J., Meyer, B.I., Peach, S., Hesselberth, J.R., Wolin, S.L., and Hammarlund, M. (2014). The RtcB RNA ligase is an essential component of the metazoan unfolded protein response. EMBO Rep. 15: 1278–1285, https://doi.org/10.15252/embr.201439531.Search in Google Scholar PubMed PubMed Central
Liao, Y., Smyth, G.K., and Shi, W. (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30: 923–930, doi:https://doi.org/10.1093/bioinformatics/btt656.10.1093/bioinformatics/btt656Search in Google Scholar PubMed
Lihanova, Y., and Weinberg, C.E. (2021). Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa. RNA Biol. 18: 1858–1866, doi:https://doi.org/10.1080/15476286.2021.1874706.Search in Google Scholar PubMed PubMed Central
Lindell, M., Romby, P., and Wagner, E.G.H. (2002). Lead(II) as a probe for investigating RNA structure in vivo. RNA 8: 534–541, https://doi.org/10.1017/s1355838201020416.Search in Google Scholar PubMed PubMed Central
Ling, M.L., Risman, S.S., Klement, J.F., McGraw, N., and McAllister, W.T. (1989). Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate. Nucleic Acids Res. 17: 1605–1618, https://doi.org/10.1093/nar/17.4.1605.Search in Google Scholar PubMed PubMed Central
Lu, C., Meyers, B.C., and Green, P.J. (2007). Construction of small RNA cDNA libraries for deep sequencing. Methods 43: 110–117, https://doi.org/10.1016/j.ymeth.2007.05.002.Search in Google Scholar PubMed
Lu, Y., Liang, F.-X., and Wang, X. (2014). A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB. Mol. Cell 55: 758–770, https://doi.org/10.1016/j.molcel.2014.06.032.Search in Google Scholar PubMed PubMed Central
Luhtala, N. and Parker, R. (2010). T2 Family ribonucleases: ancient enzymes with diverse roles. Trends Biochem. Sci. 35: 253–259, https://doi.org/10.1016/j.tibs.2010.02.002.Search in Google Scholar PubMed PubMed Central
Maughan, W.P. and Shuman, S. (2015). Characterization of 3-phosphate RNA ligase paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus highlights DNA and RNA 5-phosphate capping activity of RtcB3. J. Bacteriol. 197: 3616–3624, https://doi.org/10.1128/jb.00631-15.Search in Google Scholar
Olzog, V.J., Gärtner, C., Stadler, P.F., Fallmann, J., and Weinberg, C.E. (2021). cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2’,3’ cyclic phosphate and 5’ hydroxyl ends. RNA Biol 18: 818–831, doi:https://doi.org/10.1080/15476286.2021.1999105.10.1080/15476286.2021.1999105Search in Google Scholar PubMed PubMed Central
O'Neil, D., Glowatz, H., and Schlumpberger, M. (2013). Ribosomal RNA depletion for efficient use of RNA-seq capacity. Curr. Protoc. Mol. Biol., Chapter 4, Unit 4.19.1–188.8.131.52.1002/0471142727.mb0419s103Search in Google Scholar PubMed
Peach, S.E., York, K., and Hesselberth, J.R. (2015). Global analysis of RNA cleavage by 5-hydroxyl RNA sequencing. Nucleic Acids Res. 43: e108, https://doi.org/10.1093/nar/gkv536.Search in Google Scholar PubMed PubMed Central
Popow, J., Englert, M., Weitzer, S., Schleiffer, A., Mierzwa, B., Mechtler, K., Trowitzsch, S., Will, C.L., Lührmann, R., Söll, D, et al.. (2011). HSPC117 is the essential subunit of a human tRNA splicing ligase complex. Science 331: 760–764, https://doi.org/10.1126/science.1197847.Search in Google Scholar PubMed
Roth, A., Weinberg, Z., Chen, A.G.Y., Kim, P.B., Ames, T.D., and Breaker, R.R. (2014). A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat. Chem. Biol. 10: 56–60, https://doi.org/10.1038/nchembio.1386.Search in Google Scholar PubMed PubMed Central
Salehi-Ashtiani, K., Lupták, A., Litovchick, A., and Szostak, J.W. (2006). A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene. Science 313: 1788–1792, https://doi.org/10.1126/science.1129308.Search in Google Scholar PubMed
Sambrook, J. and Russell, D.W. (2006). Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006: 1.10.1101/pdb.prot4455Search in Google Scholar PubMed
Shigematsu, M., Kawamura, T., and Kirino, Y. (2018). Generation of 2,3-cyclic phosphate-containing RNAs as a hidden layer of the transcriptome. Front. Genet. 9: 562, https://doi.org/10.3389/fgene.2018.00562.Search in Google Scholar PubMed PubMed Central
Smith, T., Heger, A., and Sudbery, I. (2017). UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Res. 27: 491–499, https://doi.org/10.1101/gr.209601.116.Search in Google Scholar PubMed PubMed Central
Tanaka, N. and Shuman, S. (2011). RtcB is the RNA ligase component of an Escherichia coli RNA repair operon. J. Biol. Chem. 286: 7727–7731, https://doi.org/10.1074/jbc.c111.219022.Search in Google Scholar
Tanaka, N., Chakravarty, A.K., Maughan, B., and Shuman, S. (2011a). Novel mechanism of RNA repair by RtcB via sequential 2,3-cyclic phosphodiesterase and 3-phosphate/5-hydroxyl ligation reactions. J. Biol. Chem. 286: 43134–43143, https://doi.org/10.1074/jbc.m111.302133.Search in Google Scholar
Tanaka, N., Meineke, B., and Shuman, S. (2011b). RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo. J. Biol. Chem. 286: 30253–30257, https://doi.org/10.1074/jbc.c111.274597.Search in Google Scholar PubMed PubMed Central
Temmel, H., Müller, C., Sauert, M., Vesper, O., Reiss, A., Popow, J., Martinez, J., and Moll, I. (2017). The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli. Nucleic Acids Res. 45: 4708–4721, https://doi.org/10.1093/nar/gkw1018.Search in Google Scholar PubMed PubMed Central
Thompson, D.M., Lu, C., Green, P.J., and Parker, R. (2008). tRNA cleavage is a conserved response to oxidative stress in eukaryotes. RNA 14: 2095–2103, https://doi.org/10.1261/rna.1232808.Search in Google Scholar PubMed PubMed Central
Twittenhoff, C., Brandenburg, V.B., Righetti, F., Nuss, A.M., Mosig, A., Dersch, P., and Narberhaus, F. (2020). Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions. Nucleic Acids Res. 48: e71, https://doi.org/10.1093/nar/gkaa404.Search in Google Scholar PubMed PubMed Central
Weinberg, C.E. (2021). Biological roles of self-cleaving ribozymes in ribozymes. John Wiley and Sons, Ltd, Hoboken, NJ, USA, pp. 23–53, Available from: https://onlinelibrary.wiley.com/doi/10.1002/9783527814527.ch2.Search in Google Scholar
Weinberg, C.E., Weinberg, Z., and Hammann, C. (2019). Novel ribozymes: discovery, catalytic mechanisms, and the quest to understand biological function. Nucleic Acids Res. 47: 9480–9494, https://doi.org/10.1093/nar/gkz737.Search in Google Scholar PubMed PubMed Central
Weinberg, Z., Kim, P.B., Chen, T.H., Li, S., Harris, K.A., Lünse, C.E., and Breaker, R.R. (2015). New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat. Chem. Biol. 11: 606–610, https://doi.org/10.1038/nchembio.1846.Search in Google Scholar PubMed PubMed Central
Westermann, A.J., Gorski, S.A., and Vogel, J. (2012). Dual RNA-seq of pathogen and host. Nat. Rev. Microbiol. 10: 618–630, https://doi.org/10.1038/nrmicro2852.Search in Google Scholar PubMed
Zheng, L., Falschlunger, C., Huang, K., Mairhofer, E., Yuan, S., Wang, J., Patel, D.J., Micura, R., and Ren, A. (2019). Hatchet ribozyme structure and implications for cleavage mechanism. Proc. Natl. Acad. Sci. U.S.A. 116: 10783–10791, https://doi.org/10.1073/pnas.1902413116.Search in Google Scholar PubMed PubMed Central
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2021-0408).
© 2022 Walter de Gruyter GmbH, Berlin/Boston