Self-rated health (SRH) has been shown to be a stronger comprehensive predictor of health status than the clinical record. Although an association between specific pesticide exposures and health conditions has been reported in different populations, data on the relationship between pesticides exposure intensity (PEI) and SRH in greenhouse farmers is scarce. The aim of the current study was to evaluate this association among vegetable greenhouse farmers in Yinchuan City, western China.Three consecutive cross-sectional studies were conducted in the years 2015, 2016 and 2017. Face-to-face interviews by trained investigators, using questionnaires, were performed. PEI was calculated by a validated method and then categorized into high, middle and low groups. SRH was measured via a single ten-point scale question and then divided into excellent (score >5) and poor SRH (score ≤5). A multivariable logistic regression model was used to evaluate the association. Meanwhile, the dose-response and interaction effects were estimated.Pinteraction = 0.04 and 0.02, respectively); synergistic enhanced effects for poor SRH were observed.A steady association between high PEI and poor SRH  was identified. Although high PEI was significantly associated with poor SRH in males and the Han ethnicity group, no significant association was found with poor SRH in females or those of Hui ethnicity. Interaction effects of education level and frequency of breakfast with PEI were determined (These findings indicate that high PEI might be associated with poor SRH among vegetable greenhouse farmers. A lower education level and never eating breakfast contributed to an increased likelihood of poor SRH in those with high PEI. The local government should be making great efforts to promote healthy behaviors and improve protection awareness. Pesticides are widely used for the control of weeds and pests in order to expand profit from agricultural production. Studies to date have confirmed the positive association between pesticides exposure and health effects among farmers that range from acute poisoning to chronic hazards, such as mental disorder, brain tumors, 2, leukPoor self-rated health (SRH) is a useful outcome measurement and more powerful predictor of comprehensive health status than the medical record as observed by clinical physicians and could predict functional disability, mortality and morbidity–15. LinkWe conducted a population-based cross-sectional study from April to May in three consecutive years . Four large cooperative villages in Yinchuan City, the capital of Ningxia Province in Northwestern China, were chosen as the research sites. In the sample villages, most residents are engaged in vegetable greenhouse-related work. One non-repeated resident’s team  was randomly selected in each village in each survey year. Then, all of greenhouse farmers in selected team who met the requirements were invited to participate in the investigation if they were agreeable. Participants enrolled criteria were: 1. worked in plastic vegetable sheds for at least one year; 2. lived at their current address for at least five years.Information about pesticide exposures and SRH were collected via face-to-face interview by trained investigators using a questionnaire that assessed general demographic characteristics, medically diagnosed chronic diseases, pesticide spray behaviors, protection methods and hygiene habits, and SRH. Attempts to capture key missing data were supplemented at least twice by phone.Because most participants in our study were busy with farming work at the time of our investigations, verbal consent from the participating farmers was obtained prior to the interview, which helped to facilitate management and improve investigation compliance and efficiency. The design for this study was approved by the Medical Ethics Committee of Ningxia Medical University (No.2014-090).Pesticide exposure intensity (PEI) was estimated using a validated method from Dosemeci and Lee. The pesPEI = (mixing status + application method + spray behavior + whether the equipment was checked before spraying) × PPE × spillsThe score for personal protective equipment (PPE) use was obtained by asking the following questions: “What are the protective measures you take when applying pesticides?” If “never” was selected, the participant was assigned to PPE-0. If one or more of the following options were selected, PPE-1 was assigned: masks, goggles, fabric or leather gloves, old clothes; if one or more of the following options were used, then PPE-2 was assigned: gas masks, rubber boots, or clean protective clothing; and if rubber gloves were used, then PPE-3 was assigned. The PPEMixing status was defined as two-levels, never and mixed, assigned to 0 and 9, respectively.The pesticide spray application method was divided into three levels: hand spray, machine spray and mix spray; scores were 9, 3 and 6, respectively.Behaviors during the spray process were divided into five categories: drinking water, eating, smoking, chatting, and none; scores were 3, 3, 2, 1 and 0, respectively.Two options were set for whether the equipment was checked before spraying, check and never; scores were 0 and 2, respectively.The spill score was derived from a single question “Did you change your clothes after spraying pesticides?”, and the corresponding score was obtained according to four response options .Finally, we divided PEI into three groups  because of a skewed PEI distribution.SRH was measured using a single ten-point scale question: “What do you think of your health status compared with your peers?”. Respondents whose score ranged from 1 to 5 were referred to as “poor” SRH, and scores from 6 to 10 were referred to as “excellent” SRH, based on previous methods.Chronic diseases were considered due to previous literature that showed strong associations with SRH and wereChi-square tests were used to detect a difference in SRH by demographic characteristics, lifestyle characteristics, family economic status and PEI group.P value for the trend of poor SRH were calculated.A multivariable logistic regression model was used to examine the association between PEI and SRH. In the base model, only PEI was set as an independent variable. Demographic information, lifestyle variables, family economic status, number of chronic diseases and investigation year were adjusted singly or simultaneously in other models. The Odds Ratio (OR), 95% confidence intervals (CIs) and P<0.05 for all of the tests. All analyses were performed in SPSS for Windows, version 24.0 .Interaction terms were defined as family economic status, education level and breakfast frequency multiplied by PEI. The interaction effect was evaluated by terms entered into the regression model. Significance was considered at This study included 1366 participants . Of those who completed the survey, 725 were males and 641 were females. The average age was 46.84±10.27 years, ranging from 21 to 79 years. Hypertension and CHD accounted for a major proportion of chronic diseases in the current sample; 60 and 19 of them reported hypertension and CHD, respectively. Detailed sociodemographic characteristics of the participants are displayed in Of the participants, 208 (15.23%) reported poor health. A higher percentage of poor SRH were observed in high PEI . A significant distribution of poor SRH among ethnicity groups was determined, with Han higher than Hui (15.96% versus 9.55%). A low frequency of breakfast had a higher percentage of poor SRH than a regular breakfast habit. In the education groups, participants who had no formal school education had a high percentage (22.52%) of poor SRH, followed by primary school (16.74%), high school or above (10.00%) and junior high school (8.80%). In terms of family economic status, the percentage of poor SRH from Q1 to Q4 was 22.48%, 16.11%, 13.11% and 7.5%. In addition, a negative linear trend of poor SRH percentage was noted in education level and family economic status groups.The association between SRH and PEI is shown in p = 0.039) and breakfast frequency (p = 0.022) with PEI were observed. Interaction plots are shown in In addition, interaction effects of education level  and 1.581 (95% CI:1.056–2.366), respectively, from the full model. Details are presented in This study investigated the association of pesticide exposure intensity with self-rated health through validated assessment of pesticides exposure in the greenhouse vegetable farmers of western China. The results indicated a significant association between high PEI and poor SRH. The strength of association might be ensured by the fact that the results were observed in three consecutive surveys.In the current study, SRH was used to efficiently reflect the health condition of greenhouse vegetable farmers. Although SRH was defined as a subjective perception that might be limited by education experience, age, gender and occupation characteristics, 27, 28,An insignificant association was observed between medium PEI and SRH; one plausible explanation is that this was a cross-sectional study that did not have enough power to capture a small effect of long-term low-dose exposure impact on health. From the full model, a 1.5-fold increased likelihood of poor SRH was observed in those with high PEI. The neural system and mental health are affected by pesticide exposure level and high susceptibility, and a relationship between specific pesticide exposures and a physiological biochemical index, 30, 31 A higher percentage of poor SRH was observed in the Han ethnicity compared to the Hui ethnicity, which might mean that religious faith played an important mediating role. All of the Hui people are hereditarily Islamic, but lack of faith in most Han people has been reported. ReligioA linear association between education level and SRH was estimated. Farmers who had a high education level usually had a good sense of self-protection behavior that could protect their health during the spray process. A significant relationship between breakfast frequency and poor SRH was identified, which is in line with results from Jieyu Chen, whose cAn interaction effect of education level and PEI was shown as a rapidly descending trend in SRH from no formal education to high school among those in the high PEI group. A higher education level would help participants to gain knowledge of self-protection behaviors and select correct equipment when spraying pesticides. Another potential explanation might be that education had a mediating effect on adjusting mood and impacting body health. For parAlthough SRH has been shown to be obviously advantageous for predicting health status compared to clinical examination, using it to assess health status might lead to bias. The PEI level was estimated by an indirect method, which might produce more bias than an objective measurement. Other limitations of this study should be noted. First, SRH status was measured at one time-point that did not consider the change or differences in pesticide exposure by season. Second, mediating effects from potential factors in PEI and SRH were not explored. Finally, the type of pesticides and their composition were not considered, which limits our ability to explain the mechanism.This study is a reliable population-based study of the association between PEI and SRH in greenhouse vegetable farmers from western China. In conclusion, despite the above limitations, this study revealed a robust negative linear relationship between PEI and SRH from different models that adjusted for demographic, lifestyle and chronic disease. Meanwhile, the research provides visual confirmation of the interaction effects of education level and breakfast frequency with PEI. Low education and never eating breakfast could increase the odds of poor health with PEI, especially with high PEI. Apart from the causal pathways, the present multi-year cross-sectional survey implicates a needed effort by the local government to intervene in lifestyle and agricultural activities to reduce the risks associated with pesticides exposure among farmers.


The posed question arises for instance in regional gravity field modelling using weighted least-squares techniques if the gravity field functionals are synthesised from the spherical harmonic coefficients of a satellite-only global gravity model (GGM), and are used as one of the noisy datasets. The associated noise covariance matrix, appeared to be extremely ill-conditioned with a singular value spectrum that decayed gradually to zero without any noticeable gap. We analysed three methods to deal with the ill-conditioned noise covariance matrix: Tihonov regularisation of the noise covariance matrix in combination with the standard formula for the weighted least-squares estimator, a formula of the weighted least-squares estimator, which does not involve the inverse noise covariance matrix, and an estimator based on Rao’s unified theory of least-squares. Our analysis was based on a numerical experiment involving a set of height anomalies synthesised from the GGM GOCO05s, which is provided with a full noise covariance matrix. We showed that the three estimators perform similar, provided that the two regularisation parameters each method knows were chosen properly. As standard regularisation parameter choice rules do not apply here, we suggested a new parameter choice rule, and demonstrated its performance. Using this rule, we found that the differences between the three least-squares estimates were within noise. For the standard formulation of the weighted least-squares estimator with regularised noise covariance matrix, this required an exceptionally strong regularisation, much larger than one expected from the condition number of the noise covariance matrix. The preferred method is the inversion-free formulation of the weighted least-squares estimator, because of its simplicity with respect to the choice of the two regularisation parameters. In local gravity field modelling, a global gravity field model (GGM) may be considered as another noisy dataset next to the local datasets such as terrestrial and shipboard gravity anomalies, airborne gravity disturbances, radar altimeter deflections of the vertical or along-track height anomaly differences. In Stokes-based approaches, part of the information in the form of noise degree variances is routinely used for the modification of the Stokes kernel  using these data directly as observations  model. SRBFs have been used successfully in many studies of local gravity field and (quasi-) geoid modelling  grid, a terrain model for ocean and land with a spatial resolution of The following experimental setup was chosen. The disturbing potential was set equal to the regularised GOCO05s spherical harmonic model from degree 151–200. The area of interest was bounded by We selected a number of Reuter grids with different control parameters ranging from N. For each N, the statistics refer to a least-squares solution obtained for a depth of the Poisson wavelets and a regularisation parameter providing the smallest model error among a set of candidate depths and regularisation parameters. As expected, the model error decreases with increasing point density. What model error is acceptable depends on the impact of data noise on the estimated quasi-geoid model. An indication of the latter is obtained when propagating the GOCO05s noise covariance matrix into height anomalies. For GOCO05s complete to degree 200, the height anomaly noise standard deviations range from 1.6  to 2.7 cm over the area of interest. The maximum absolute model error should be significantly smaller than 1.6 cm. Table Table The condition number of the height anomaly noise covariance matrix for data on a Reuter grid with mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimNote that the model error depends on various settings such as the type of the SRBF, the grid used to locate the poles of the SRBFs and the data points, respectively, the area of interest, the extension of the parameterisation area beyond the data area, etc. Hence, each choice may lead to a different model error as function of the data point density.In our experiments, the parameterisation area was chosen identical to the data area. Several studies suggest to extend the parameterisation area beyond the data area  apply Tikhonov regularisation Tikhonov  to the iE is the expectation operator and D is the dispersion operator, L is the degree up to which the GGM is used when synthesising the data, R, l. For the Poisson wavelets of order 3, it is The functional model of the GGM dataset is written as a linear Gauss–Markov model,n is the number of columns and rows of the matrix n singular values Suppose the singular value decomposition (SVD) of the cofactor matrix mentclass2pt{minimWhen using Tikhonov regularisation directly applied to the cofactor matrix, we need to find a suitable value of the regularisation parameter n is the number of observations, m is the number of parameters, and According to Grafarend and Schaffrin , there imentclasspt{minimaAnother approach to deal with an ill-conditioned noise covariance matrix is offered by Rao’s unified theory of least-squares Rao , 1978. Tg to Rao , 1978, tIn the numerical experiments of Sect. r is identical to the rank of Note that the g-inverse nt}Q Rao . In the q, and compute the g-inverse q largest singular values of In this study, we test a whole range of truncation indices The estimators of Sects. The performance of the afore-mentioned methods were investigated using numerical experiments. We used the experimental setup of Sect. From every least-squares estimate entclass1pt{minimaWe compute the dispersion To investigate the quality of the weighted least-squares solutions, we use two different measures.r of Eq. , which is defined asR is the resolution matrix (i.e. the matrix n is the number of parameters (e.g. Aster et al. RD is an indication of the contribution of the data to the estimated SRBF coefficients in the presence of regularisation; the larger R, the higher the contribution of the data to the estimated SRBF coefficients. According to Table Table T of Eq. , which iFigure  cf. Fig  and the  cf. Fig , respectFigure q. A clear minimum is attained at The results for the GLS estimate are shown in Fig κ of Eq.  as functsing Eq.  may be pTable  entclass1pt{minimaFigure In this study, we investigated three methods to deal with the high condition number of the noise covariance matrix of a state-of-the-art GGM after propagation into gravity field functionals over a local data area. This problem is relevant when estimating a local model of the disturbing potential considering all available datasets as being noisy.We showed that the noise covariance matrix of height anomalies propagated from the full noise covariance matrix of GOCO05s, has a gradually decreasing singular value spectrum without any noticeable gap and a high condition number. The latter depends among others on the density of the points where the height anomalies are synthesised. The density has to be chosen high enough to guarantee that the error of the local SRBF model of the disturbing potential is negligible compared to the effect of the noise in the data. We showed that this requires a point density, which is higher than the maximum degree of the GGM suggests. The associated height anomaly noise covariance matrix had a condition number larger than the reciprocal value of the relative rounding error unit of IEEE 754 double precision arithmetic. Therefore, a straightforward computation of the weight matrix would imply the loss of all significant digits.To deal with the high condition number of the noise covariance matrix, we investigated three methods: Tikhonov regularisation of the noise covariance matrix in combination with the standard formula of the weighted least-squares estimator, an alternative formula of the weighted least-squares estimator, which does not require to compute the inverse of the noise covariance matrix, and Rao’s generalised least-squares estimator. Our experiments indicate that these methods and the main findings of Sect. We showed that all three methods provide least-squares estimates of the SRBF coefficients which were identical within noise. Moreover, the dispersion matrices of the estimated SRBF coefficients and of height anomalies at a set of control points agreed very well with each other and with the height anomaly noise covariance matrix directly propagated form the full noise covariance matrix of spherical harmonic coefficients of the GGM. Prerequisite was that the two regularisation parameters each of the three methods knows, were chosen properly. We demonstrated that a parameter choice rule which uses the dispersion matrix of height anomalies at a control dataset allowed such a choice.Among the three methods, we prefer the inversion-free weighted least-squares estimator. We showed that the choice of the two regularisation parameters can be reduced successfully to the choice of a single parameter, and the least-squares estimate and its dispersion matrix appeared to be quite robust against this choice in the numerical experiments. The fact that the inversion-free weighted least-squares estimator requires the solution of a system of linear equations of a size equal to the number of data does not pose numerical problems in real applications. We found that the weak point of Rao’s generalised least-squares estimator is the need to compute a g-inverse of a matrix with a gradually decreasing singular value spectrum without any noticeable gap. In this study, we computed such a g-inverse successfully with a truncated singular value decomposition. However, whether this applies to other situations than considered in this study, is an open question. Using Tikhonov regularisation of the data noise covariance matrix in combination with the standard formula for the weighted least-squares estimator, required an exceptionally strong regularisation of the data noise covariance matrix to obtain a good least-squares estimate of the SRBF coefficients and an accurate dispersion matrix of the estimated SRBF coefficients. We found that the major drawback of this method is the need to search a two-dimensional space to find optimal values of the two regularisation parameters.


Starting from existing approaches, we outline particular challenges and opportunities for future circRNA-based therapeutic approaches that exploit stability and molecular effector functions of native circRNAs. We end with considerations which designer functions could be engineered into artificial therapeutic circular RNAs.It has recently been reported that thousands of covalently linked circular RNAs (circRNAs) are expressed from human genomes. circRNAs emerge during RNA splicing. circRNAs are circularized in a reaction termed “backsplicing,” whereby the spliceosome fuses a splice donor site in a downstream exon to a splice acceptor site in an upstream exon. Although a young field of research, first studies indicate that backsplicing is not an erroneous reaction of the spliceosome. Instead, circRNAs are produced in cells with high cell-type specificity and can exert biologically meaningful and specific functions. These observations and the finding that circRNAs are stable against exonucleolytic decay are raising the question whether circRNAs may be relevant as therapeutic agents and targets. In this review, we start out with a short introduction into classification, biogenesis and general molecular mechanisms of circRNAs. We then describe reports, where manipulating circRNA abundance has been shown to have therapeutic value in animal disease models  High-throughput RNA-sequencing has shown that at least 20% of presently active genes express circRNAs . CircRNADetails on circRNA biogenesis are described in recent reviews . In thisSplicing not only makes the linear transcriptome more diverse, but splicing also creates a very diverse set of circular RNAs. There is a tendency toward higher circRNA formation in more highly expressed genes, but overall, circRNAs are produced cell-type specifically  and indeAnalyses in different animal models have consistently shown that forward splicing is dominant, but backsplicing frequency is favored depending on context and availability of splice sites : Two funThe following parameters determine circRNA biogenesis :(A)Reverse complementary repeats in the flanking introns, for example of the family of Alu repeats in the human genome, favor the backsplice reaction by hybridizing with each other and bringing splice sites in close proximity .(B)A rather specialized compendium of splicing factor and RNA-binding proteins partakes in backsplicing through positioning splice sites in 3-dimensional space in the nucleus . This in(C)The efficiency of backsplicing increases when forward splicing was kinetically slowed-down, such as after linear mRNA polyadenylation was impaired and read-through into adjacent genes occurred, or after core-spliceosomal components, such as the SF3a/b complexes, were limited in promoting the forward splicing reaction .Figure 1A)  .Details of known circRNA-effector mechanisms and functions in physiology and disease are described in recent reviews . Briefly(1)circEIF3J and circPAIP2 associated with promoters depending on the U1 non-coding RNA (Circular RNAs may influence RNA polymerase II (RNAP II) initiation and elongation of their host gene. As best understood cases, subgroups of EIciRNAs  and ciRNding RNA , which i(2)circMbl CDR1as and Sry, carry a higher density of microRNA binding sites. These sequester and functionally inactivate microRNAs, an effect termed “sponging” circMBL binding to MBL protein, circPOLR2A, circDHX34 binding to NF90 and NF110, circANRIL binding to PES1 and circPAPBN1 binding to HuR Drosophila circMbl, circPde8, circTai, circCdi and mouse circZNF609, these 5′UTRs structurally folded into internal ribosome entry sites (IRES). This allowed the circRNAs to associate with the small subunit of ribosomes and become templates for protein translation despite being circular .The molecular analysis of circRNAs is still in its infancy, but it is becoming clear that circRNAs can be functionally relevant, and that functions can be quite diverse. In the following chapter, we will focus on those circRNAs whose therapeutic potential can be assessed from knockdown or circRNA overexpression in mouse disease models of CVD MFACR  was identified to be upregulated in a mouse model of heart ischemia/reperfusion .The mouse circRNA erfusion . MFACR wfunction . Thus, aHRCR (acronym for heart-related circRNA) was found to be downregulated during experimentally-induced cardiac hypertrophy  and protects from hypertrophy in injured hearts. Systemic HRCR overexpression in a mouse heart failure model inactivated miR-223, hence, activating its target ARC. This also curbed cardiac hypertrophy. Since human miR-223 levels were increased and ARC levels decreased in failing human hearts, a therapeutic option would be to deliver HRCR or to inhibit its downregulation (Table 1).The mouse circRNA ertrophy . Having circ-Amotl1 was enriched in the neonatal (regenerative) heart compared with mature hearts in humans . Whether the therapeutic effect in the rodent disease model is due to the physical interaction of human circ-Amotl1 with the mouse AKT-PDK1 complex is still not fully clear. It should also be noted in this context, that human circ-Amotl1 is not conserved in mouse or rat, although triggering consistent cellular effects. At least theoretically one can expect that the structural conservation of the AKT and PDK1 proteins are sufficiently high in mammals to allow trans-species interaction of circRNA with these proteins, but this remains to be shown.n humans . Systemihy model . As an eorigenic  .Chemotherapeutic drugs and other stressors trigger senescence-associated pathological changes in hearts, and  circRNA . When siecreased . Conversnhibitor . Also, FcircHIPK3 is induced in diabetic human retinas . retinas . When syll types . Additioer cells , and imper cells . TherefocircZNF609, a proposed miR-615-5p sponge, was shown to be induced in diabetic human retinas. circZNF609 contains one seed-matched binding site. The mouse orthologous circRNA exerted deleterious cell overproliferation and a pro-angiogenic role while expressing shRNAs against cZNF609 reduced capillary degeneration and pathological angiogenesis in a mouse oxygen-induced retinopathy model .hy model . The accntiation , raisingcircDLGAP4, a proposed miR-143 sponge, was less abundant in brain tissue in a rodent cerebral stroke model, and in blood plasma of human acute ischemic stroke patients .patients . The cirThe studies described here are representative of how circRNAs are currently used as therapeutic agents and targets in different pathologies. It should be understood that the presented cases are published studies related to CVD, and not necessarily those with the highest expectable therapeutic potential. In all cases, circRNAs and circRNA-knockdown construct are delivered systemically, although lesions are local, and although the candidate circRNAs are known to be endogenously expressed in more than one organ. It is, therefore, rather astonishing, but also promising, that such interventions were able to ameliorate specific CVD endpoints at all. It must be stated that before clinical trials could be thought of, the expected negative effects on proliferation and differentiation of non-target cells and organs have to be addressed.in vivo. A single study, so far, reported on the stability of synthetic circRNAs in cultured cells in vitro .A future therapeutic use of circRNAs could be envisioned in two ways. One is the modulation of native disease-linked circRNAs by therapeutic knockdown or by ectopic expression. A second is the engineering of non-native  circRNAs with designer molecular effects  and (2) Engineering of in vitro-produced circRNAs . To modulate native circRNAs, one can overexpress native protective circRNAs and reconstitute missing circRNAs from genetic vectors , deplete endogenous disease-promoting circRNAs , or correct aberrantly produced circRNA-isoforms . On the other hand, in vitro-produced RNA circles can be transferred into cells as unmodified RNA  or as modified RNA .At least two concepts for therapeutic modulation of endogenous disease-linked circRNAs are conceivable: (1) Modulation of native circRNAs . These encode a mini-gene cassette with exon(s), endogenous splice donor and acceptor sites, and flanking intronic inverted repeats that support RNA backfolding . Alternatively, and independently of the cellular spliceosome, circRNAs are successfully expressed also from within intron boundaries encoded from the anticodon stem-loop on an engineered tRNA construct . In-cell production of human circRNAs can be engineered to involve RNA ribozymes that mediate circularization by RNA self-processing . Another tangible approach for post-transcriptional production of circRNAs would be to use ASOs that bind to splice sites or splice enhancers and increase the frequency of co-transcriptional backsplicing. This would enhance the primary alternative splicing event so that more circularization can occur when more exon-containing lariats are present . Given the competition with linear splicing  of the total mRNA output of any protein-coding gene  . In the future, modern ssRNA-targeting by CRISPR-Cas9/13 variants . To achieve specificity in circRNA-knockdown, all these nucleases must be guided selectively to the circRNA-specific backsplice-junction  have been depleted in vivo using short hairping (shRNAs) and small interfering siRNAs to trigger RNAi. An already implemented and very specific therapeutic approach for circRNA knockdown relates to depletion of fusion-circRNAs (f-circRNAs): These arise when chromosomal translocations bring introns from two unrelated genes in close genomic vicinity, which undergo backsplicing . This has not yet been experimentally tested. As mentioned before, inhibition of backfolding should in theory block circularization, and in some instances even favor linear mRNA splicing, given the competition between co-transcriptional backsplicing and mRNA maturation.Depletion of endogenous disease-promoting circRNAs has already been achieved using standard genetic tools, RNA interference (RNAi) or ASO-mediated RNase H-dependent degradation : Fusion of catalytically-inactive Cas13 enzyme with an ADAR deaminase has already been proven successful for manipulating linear RNA sequence at the level of the transcript . Different methods exist to achieve RNA circularization  and to formulate such circRNAs, such as by coating them with native RNA-binding proteins , or by adding chemical moieties that change the physicochemical properties of the RNA . While the consequences of artificial circularizing linkers have to be addressed functionally in each case, first estimation allow to predict that their structural and chemical alterations tend to be minor . Among different circularization strategies, methods using ribozymes and click-chemistry-based linkage seem to be particularly efficient and cost-effective alternatives to commercial recombinant RNA ligases.Delivering synthetic RNA circles into cells is another therapeutic approach. Such synthetic circRNAs are produced 5′ bonds  or by tr5′ bonds ] . Here, we will discuss several major concepts for engineering designer circRNAs, with the most feasible ones ranked first: (A) MicroRNA-sponging by circRNAs; (B) Circularization of known linear therapeutic RNAs; (C) Protein translation from circRNAs; (D) Modulation of the immune system by circRNAs; (E) Protein activity control by circular RNA aptamers; (F) Transcription/splicing control by circRNAs; and (G) Engineering autonomous RNA replication of therapeutically delivered circRNAs in vivo.Aside from modulating the expression of native circRNAs, circRNAs can also be engineered to carry specific properties ((A)Figure 3A). Considering recently published experiments, only a few endogenous circRNAs like circCDR1as or circSry  that targets the virus by complementary binding to miR-122 Figure 3B). This is possible, because compared to the rigid dsDNA helix . As described above, more than 99% of endogenous circRNAs are never productively associating with ribosomes, and in no case, so far, has the function of a circRNA-templated polypeptide been documented. Therapeutically it is, however conceivable, that exons encoded in engineered circRNAs could be translated to proteins when the ribosome is capture via an IRES, and when a complete open reading frame with stop and start codon were included through engineering . Such a strategy could be interesting for many linear RNAs/mRNAs that are already therapeutically used, for gene expression control, vaccination, or as aptamers (see existing reviews for example of linear therapeutic RNAs (Anabaena) pre-tRNA as ribo-enzymatic factor. Translation was particularly strong when using unconventional IRES elements, such as from the Coxsackievirus B3, and when adding structural spacing elements between IRES and splice sites. Comparing to state-of-the-art translation from transfected linear mRNAs with 5′Cap, 3′polyA tail and internal 5′methoxyuridine modifications for stabilized expressed, translation from unmodified circRNAs drove 50% more protein and provided protein over a two-times longer time period (80–120 h after a single RNA transfection)  .(C)Figure 3C).Care has to be taken about the specificity of translation from circRNAs. In the absence of in-frame stop codons and termination signals, or after read-through, rolling-circle-translation can occur on circular RNAs. Rolling-circle translation can lead to translation of multimeric repetitive protein motifs, which could be useful for cell engineering, but which can become toxic for cells if happening in an uncontrolled fashion  Figure 3D). In this pathway, RNA-sensing in the cytoplasm triggers innate immunity, and innate immune signaling is known to contribute to adaptive immunity by diverse routes. One option would, thus, be to use circRNAs as tools to purposefully boost innate immune signaling to counteract disease-induced immunosuppression, or as adjuvants for boosting adaptive immunity during vaccination. This is not far-fetched as circularity of RNA has recently been shown to be sensed by the RNA-binding RIG-I molecules, which trigger downstream innate immune signaling Figure 3E). Among many other options, aptamers are useful for selective drug delivery or protein activity control. Circularizing them might make them more potent. circRNAs have already before been suggested to be potentially useful to affect the availability and subcellular sorting of RNA-binding proteins RBPs  has been devised as a powerful unbiased PCR-based method for molecularly improving binding or effector mechanism in vitro for linear aptamer nucleic acids  trans-regulatory circular RNAs exist, and these spur the hope to be able to exploit this mechanism for therapeutic purposes : For example, the class of intron-only ciRNA is thought to influence genomic loci in trans, whereby ciRNAs bind (RNAP II) and boost transcription .Many endogenously expressed 3′-5′-linked circRNAs are thought to be modulators of transcription and splicing of linear mRNA, mostly of cognate linear transcripts produced from the circRNA host gene. The modulation happens when backsplicing occurs cotranscriptionally within pre-mRNA . Specificription . Indepenin trans b. It was(G)Figure 3G). For example circular RNA viroids in plants are replicated as RNAs in vivo. This occurs by an RNA-rolling-circle mechanism, whereby host DNA-dependent RNA-polymerase is repurposed for transcription of the RNA circle , cannot use end-modification in a circular RNA. Instead such modifications would have to be introduced, for example, by functionalizing the ribose 2′ hydroxyl group in the circRNA backbone. A lot of work has been dedicated to achieve tropism in atherosclerosis therapy by using homing peptides or fatty acids, or functionalized RNAs and lipid carriers that target only atherosclerotic plaques  to passively pass the cell membrane’s phospholipid bilayer . InsteadAs with any therapy, it is anticipated that modulation of circRNAs might also have side effects. The knockdown of circRNAs suffers from the same potential off-targeting effects as for linear mRNA interference, aggravated by the fact, that knockdown must specifically target the unique circRNA back-splice junction and is, thus, even more, constrained . Also, tTable 1, it has not yet been explored, whether the therapeutic effect was due to the change in circRNA level in the diseased target tissue. Especially for CVD, disease parameters might change as a consequence of the systemic effects of circRNAs on liver function, metabolism or the immune system. In this case, superior methods may deliver the therapeutic circRNAs more directly to those primary effector organs and increase the therapeutic potency.It should also be noted that for all cases of therapeutic circRNAs described in in vitro synthesized formulations. As of now, it is still difficult to synthesize circRNAs at a sufficient scale in a cost-effective manner. In vitro synthesis of circRNAs is currently still performed with recombinant enzymes, which is costly when substantial amounts are required for therapy. Thus, alternatives for routine high-scale synthesis are being developed, such as allosterically regulated ribozymes, which perform circularization but are unable to re-cleave the circular RNA end-product and, thus, are envisioned to increase circRNA yield (Form today’s perspective, it is well conceivable that circRNAs may serve as therapeutic agents and targets in the near future. CircRNAs might either be modulated intracellularly or administered as NA yield . DiffereNA yield . This isNA yield . If and Overall, it has taken 4 decades to overcome some of the major hurdles for using linear RNA as therapeutics. Since technological insight is in many cases directly transferrable from linear therapeutic RNAs to circRNAs, the use of circRNAs as therapeutic agents and targets in human diseases is only a question of time and might occur earlier than expected.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arthrobacter luteus (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements–enhanced cross-linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome-wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA-binding protein, strengthening the model of TEs as functional modules in lncRNAs.—Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C-terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)–enhancing translation of sense protein-coding mRNAs. By using RNA-interacting domainome technology, we identify the IL enhancer-binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA-binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right  Prev complex . Binding complex , 22. Fur complex , 24. By  complex .One of the key features of genomes’ organization is that most genes share their genomic region with another gene on the opposite filament, forming sense-AS (S/AS) pairs , 26. AlmWe previously showed that the mouse lncRNA AS ubiquitin C-terminal hydrolase L1 (Uchl1) can enhance translation of sense protein-coding Uchl1 mRNA through the activity of an embedded TE of the short interspersed nuclear element (SINE) B2 type . AS Uchl31in vitro  and with functional assays in cell culture.Here, we identify proteins that interact with the invSINEB2 of AS Uchl1. To this end, we employ RNA-interacting domainome (RIDome), a high-throughput interaction discovery platform that combines the selection of a phage cDNA library displaying filtered open reading frames (ORFs) with next-generation sequencing (NGS)  outline. In brieWe find that the dsRBP IL enhancer-binding factor 3 (ILF3) is an interacting partner of AS Uchl1. This interaction specifically requires one of the 2 dsRNA-binding motifs (dsRBMs) of ILF3 and the invSINEB2 sequence in AS Uchl1. ILF3 also binds FRAM sequences, the embedded TEs in human SINEUPs. By bioinformatics analysis of enhanced CLIP (eCLIP) data for ILF3 from the Encyclopedia of DNA Elements (ENCODE), we confirm that this RBP is a major interacting protein of Alu sequences in human. In addition, we also demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3.et al. (Plasmids expressing AS Uchl1 full-length (FL), AS Uchl1 ΔB2, AS Uchl1 ΔAlu, and AS Uchl1 ΔTE (previously referred to as ΔTOT) were prepared as previously described in Carrieri et al. .in vitro transcription . Template DNAs were prepared by PCR using specific primer pairs in which the forward oligonucleotide was tailed with a T7 RNA polymerase minimal promoter. Synthesized RNAs were analyzed by electrophoresis, purified , quantified by spectrophotometry, and biotinylated at the 3′ end . RNA samples were stored at −80°C until use.RNA baits used for biopanning experiments and successive ELISA-based assays were synthesized by 11 colony-forming units per microliter, and for each selection we used 1012 phages. Selections were done using 2 SINEUP-related RNA baits  were washed in 10 mM Tris HCl pH 8.0, 1 mM EDTA, 250 mM NaCl, 0.5% Triton X-100 (TENT buffer) and then incubated with 10Escherichia coli. The beads were washed 3 times in TENT buffer; then phages from the precleared libraries were added to the RNA-conjugated beads and incubated for 45 min at room temperature in the presence of 1 μg/μl of ssDNA or tRNA. Beads were then washed extensively in TENT buffer. Bound phages were eluted by a treatment with RNAse A  for 2 min at room temperature; then the supernatant containing the phages released from beads was used to infect 2 ml of E. coli DH5α for 45 min at 37°C. The eluted phage pool was amplified in DH5α cells, and the procedure was repeated for a second round of selection; the stringency of selection was enhanced by increasing the number of washing steps. To avoid an excessive restriction in the output diversity, only 2 cycles of selection were performed for all protocols. After the second round of selection, colonies growing on agar plates were harvested, and plasmid DNA was isolated by standard miniprep procedure. cDNA inserts were PCR-amplified with barcoded molecular identifier–tagged primers and sequenced with an Illumina SmartSeq platform .For biopanning experiments, the RNA baits were diluted to 30 nM in TENT buffer containing 100 U/μl of the RNAse inhibitor Superase-In (Thermo Fisher Scientific); then 100 μl (3 pmol) were added to 20 μl of streptavidin magnetic beads and incubated for 20 min at room temperature. Selections were performed using 2 protocols that differ in the competitor used: single-stranded DNA (ssDNA) from herring sperm or tRNA from https://www.ngs-trex.disit.unipmn.it/Trex/cms/) as previously described .Sequences were processed with the NGS Transcriptome Profile Explorer (NGS-Trex) system  and K562 cell lines (https://www.encodeproject.org/experiments/ENCSR438KWZ/). For each cell line 2 replicate experiments were performed. We downloaded the following inputs : ENCFF340GPD (https://www.encodeproject.org/files/ENCFF340GPD/), ENCFF841BJF (https://www.encodeproject.org/files/ENCFF841BJF/), ENCFF353RQP (https://www.encodeproject.org/files/ENCFF353RQP/), and ENCFF623LPT (https://www.encodeproject.org/files/ENCFF623LPT/).Human eCLIP data for ILF3 were downloaded from the ENCODE project –42 for Hhttps://useast.ensembl.org/index.html) and were relative to the Ensembl v.83. Repetitive element annotations relative to the GRCh38 assembly were obtained from the RepeatMasker (http://www.repeatmasker.org/).The files contain locations of peaks associated to ILF3 bindings mapped on the human genome (assembly GRCh38) and their enrichment with respect to the input. Peaks were annotated, also keeping in account the strand. Information on the protocols and methods used to produce these data is openly available on the ENCODE project website. Human gene annotations (assembly GRCh38) in GFF3 format were downloaded from Ensembl   fusion products, ORF fragments were excised from the phagemid DNA with the restriction endonucleases PteI and NheI (Thermo Fischer Scientific), subcloned into a custom-designed pGEX-Flag expression vector  cells. Bacterial cultures (100 ml) were grown at 28°C until optical density at 600 nm reached 0.5 and then induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG)for 3 h. Bacteria were collected by centrifugation, and pellets were suspended in lysis buffer , incubated at 4°C for 30 min, and sonicated for 2–3 min. Cell debris was removed by centrifugation and supernatants combined with glutathione-agarose beads  at 4°C for 60 min under gentle rotation. After 3 washes in PBS–Tween 0.1% followed by 3 more washes in PBS, GST fusion proteins were eluted in elution buffer . Proteins were dialyzed against PBS and checked for purity and concentration by SDS-PAGE. Quantitative densitometry of Coomassie Blue–stained proteins was calculated with ImageJ software    using boet al.  as previously described in Patrucco et al. . The bioGST-dsRBM2 was serially diluted in running buffer to the concentrations 300–3.7 nM and injected at 25°C at a flow rate of 30 µl/min for 2 min. Analysis were performed in duplicate, and any background signal from a streptavidin-only reference flow cell was subtracted from every data set.Human embryonic kidney (HEK) 293T/17 cells were obtained from American Type Culture Collection (ATCC-CRL-11268) and cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum , penicillin, and streptomycin.6 HEK 293T/17 cells were plated in 10-cm dishes and transfected with AS Uchl1 FL plasmid using FuGene HD Transfection Reagent , following the manufacturer’s instructions. RNA and proteins were extracted from the same transfection in each replica.For RNA immunorecipitation (RNA-IP) experiments, 2.5 × 105 cells were plated in 6-well plates and transfected with AS Uchl1 FL, AS Uchl1 ΔB2, AS Uchl1 ΔAlu, or AS Uchl1 ΔTE.For nucleocytoplasmic fractionation experiments, 4 × 102, 0.5 mM DTT, protease inhibitors, and 20 U/ml Superase RNA inhibitors). Coating with antibody or control IgG was carried out by overnight incubation of blocked beads with 20 μg of anti-ILF3 antibody  or 20 μg of mouse IgG (as a control) in a final volume of 180 μl. Lysis was performed using 1 ml RIP lysis buffer. Lysates were solubilized by sonication with 2 short pulses (15 s). Between the 2 cycles, samples were kept on ice for at least 2 min. Insoluble material was removed by centrifugation at 13,000 rpm for 10 min. Total lysate was precleared via incubation with 100 μl of uncoated blocked beads for 30 min at 4°C with gentle rotation. After recovery from beads, total lysate was split and incubated with specific antibody or control IgG-coated beads overnight on a rotary platform at 4°C. One-twentieth of total precleared lysate was kept before splitting as immunoprecipitation input. Bead-antibody-lysate complexes were washed 6 times  in a cold room. For reversal of cross-linking and elution, beads containing the immunoprecipitation samples were resuspended in 100 μl of elution buffer  and incubated at 70°C for 45 min. Supernatants were recovered and resuspended in 1 ml of Trizol (Thermo Fisher Scientific), and both RNA and proteins were extracted according to the manufacturer’s instructions.Stock solutions were prepared with RNase-free water (treated with diethylpyrocarbonate). Lysis and wash buffers were prepared fresh and kept on ice; all steps, including centrifugation, were performed at 4°C. Forty-eight hours following transfection, cells were washed with PBS, collected by gentle scraping, and centrifuged. Pellets were washed twice with PBS, and cells were fixed in 1% formaldehyde  in PBS for 10 min at room temperature with slow mixing and then quenched in 0.25 M glycine (pH 7) at room temperature for 5 min. Cells were subsequently harvested by centrifugation at 3000 rpm for 4 min and washed twice with ice-cold PBS. One hundred microliters of sheep anti-mouse magnetic beads  were washed 3 times in washing buffer , blocked with 3 washes in 0.5% BSA, and finally washed twice in RIP lysis buffer  according to the manufacturer’s instructions. RNA was eluted and treated with Turbo DNA-Free Kit (Thermo Fisher Scientific) for 15 min at 37°C to avoid plasmid DNA contamination. RNA quality was finally checked on a formaldehyde agarose gel.cDNA was prepared from 250 ng of purified RNA using iScript cDNA Synthesis Kit  according to the manufacturer’s instructions. For RNA-IP experiments, equal volumes of DNAse-treated RNA samples were used for reverse transcription. To monitor the efficiency of DNAse treatment, an equal amount of each RNA sample was retrotranscribed in the absence of reverse transcriptase.Real-time quantitative PCR reaction was performed on diluted cDNA (1:2.5) using Sybr-Green PCR Master Mix (Bio-Rad) and an iCycler IQ Real-Time PCR System (Bio-Rad). In RNA-IP experiments, undiluted cDNA was used as real-time quantitative PCR input.CytB) gene was amplified using the forward primer, 5′-CAATGGCGCCTCAATATTCT-3′, and the reverse primer, 5′-AATGTATGGGTGGCGGATA-3′. Amplified transcripts were quantified using the comparative tC method, and relative gene expression was calculated with the ΔΔtC method  and AS Uchl1, ubiquitin C (UBC), and precursor rRNA (pre-rRNA) were as previously described  and 53. t method .For Western blot analysis, cell pellets were directly dissolved in Laemmli sample buffer. For RNA-IP experiments, ILF3 immunoprecipitation efficiency was monitored by loading the whole fraction of proteins recovered from the organic phase after Trizol extraction, following resuspension in Laemmli sample buffer. All lysates were briefly sonicated, boiled, and loaded on 10% polyacrylamide gels. Immunoblotting was performed with the following primary antibodies: anti-ILF3 , 1:500 overnight, and anti–β-actin , 1:2000. Signals were revealed after incubation with HRP secondary antibodies  1:1000 for 1 h at room temperature, in combination with ECL . Image detection was performed with Alliance LD2-77WL system . Image quantification was done using ImageJ software.Nucleocytoplasmic fractionation was performed as previously described in ref. 5) were harvested on a 6-well plate and cotransfected with 4 μg of AS Uchl1 plasmid and 4 μg of ILF3 small interfering RNA (siRNA)  or control siRNA  with 10 μl of Lipofectamine 2000 (Thermo Fisher Scientific) in serum-free DMEM with no antibiotics. After 24 h, a second round of transfection was performed, using 2 μg of both plasmid and siRNA. On the following day, medium was changed with 10% FBS-DMEM. At 48 h from the second transfection, cells were collected for fractionation. One-twentieth of the total cells was suspended in Laemmli sample buffer for Western blot analysis of ILF3 protein levels in silenced and control cells. Nucleocytoplasmic fractionation was performed as previously described, and cell fractions were suspended in 1 ml of Trizol.HEK 293T/17 cells  1:50 in blocking solution, washed in PBS 3 times, and finally stained with AlexaFluor 488– or AlexaFluor 594–labeled anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher Scientific), 1:250 in blocking buffer. Nuclei were visualized with DAPI (1 μg/ml). Anti–DJ-1 1:250  was usedsd for n ≥ 3 replicas. Statistical analysis was performed using Excel software. Statistically significant differences were assessed by a Student’s t test. Values of P < 0.05 were considered significant.All data are expressed as means ± To identify proteins that interact with natural SINEUP lncRNAs, we employed RIDome . The typE. coli. With respect to canonical FL cDNA phage libraries, this approach has the advantage of generating a normalized library of protein domains (the Domainome) that are homogeneous in terms of peptide length and sequence coverage. It is noteworthy that despite the fact the library derives from only 3 human organs, almost all annotated RBPs and transcription factors are represented by at least 1 read (etc.), regardless of its tissue or organism of origin. Selections were performed using two 3′-biotinylated, in vitro–transcribed RNA baits  and hnRNPA3, 2 known RBPs that were not enriched during the library selection. The phage expressing ILF3-dsRBM2 generated a strong signal on invSINEB2 compared with the negative control , whereas the binding of SRSF5 and hnRNPA3 to invSINEB2 was negligible C. We nexIn vitro biotinylated invSINEB2 RNA was immobilized on a streptavidin-coated sensor chip analyzed on a Biacore T100, as described in Materials and Methods. The resulting sensorgram from invSINEB2-ILF3 interaction analysis did not totally adjust to a 1:1 binding model, as shown in Ka), dissociation rate (kd), and equilibrium dissociation constant or affinity constant Kd were calculated for the invSINEB2 RNA–ILF3 interaction after adjustment to this 1:1 binding model. A Kd of around 94.90 nM was calculated for this interaction, with association (Ka) and dissociation rate (kd) constants equal to 3.84 × 104 (M/s) and 3.64 × 103 (s), respectively.To further characterize the binding kinetics of the ILF3 mouse-human dsRBM2 to the invSINEB2, we used SPR. in vitro.In summary, these results support a direct binding between the mouse invSINEB2 and the mouse-human ILF3 dsRBM2, which provides the specific domain mediating the interaction with AS Uchl1 baits vs. control was quantified by real-time quantitative PCR and normalized to the mRNA level of the housekeeping gene UBC, previously described in ref. To validate and study AS Uchl1–ILF3 interaction in cells, we used the FL cDNA clone for AS Uchl1 (AS Uchl1-FL)  and carrin vitro and upon ectopic expression to ILF3.Recently, R12A-AS1, NAT to human protein phosphatase 1 regulatory subunit 12A, has been shown to be the representative transcript for human natural SINEUPs. Its activity is mediated by an embedded FRAM acting as ED. FRAM, a human TE, supports SINEUP function when transferred to a chimeric AS RNA with BD that is AS to the mRNA of interest, including the 1 encoding for the green fluorescent protein (GFP) and called hminiSINEUP-GFP   in HEK 293T/17 cells. As shown in P < 0.05 in both replicates. Considering the general mapping on the genome, more than 85%  of these peaks resulted in overlap with repeated elements. Randomization analysis demonstrated that SINEs are by far the most enriched TE  of the total peaks were in overlap with at least 1 genic region. Of these almost 96%  overlapped a coding gene, whereas 4% (564) overlapped a noncoding one. Most of genic overlaps, with respect to current annotation, were associated with introns. Indeed, only 7% of the coding genic peaks (951 peaks in 443 genes) and 16% of the noncoding ones (90 peaks in 36 genes) were exonic. The majority of exonic overlap was concordant with the strand of transcription (98% for coding and 93% for noncoding peaks).When we then considered the association of ILF3 peaks overlapping SINEs embedded in annotated exons, we obtained a total of 304 peaks overlapping SINEs in exons from 172 coding genes and 48 peaks overlapping SINEs in 23 noncoding genes. In coding genes, 38% (118) of peaks overlapped with embedded direct SINEs, whereas 60% (183) overlapped with embedded inverted SINEs. In noncoding genes, 25%  of peaksUsing independent methodology, these results confirm ILF3 binding to SINEs embedded in coding and noncoding genes. In addition, the data demonstrate that ILF3 binds with a statistically significant preference for inverted elements.Because embedded TEs have been recently associated to nuclear localization of lncRNAs , we inveAdjacent to the 3′ of the SINEB2 sequence, another TE, a partial Alu repeat, is present in the AS Uchl1 third exon . When anWe finally investigated the effects of combined removal of the invSINEB2 and Alu elements B on RNA To assess whether ILF3 may regulate AS Uchl1 RNA nuclear localization, we first carried out immunofluorescence analysis showing that endogenous ILF3 localizes in the nucleus of HEK 293T/17 cells, with no relevant signal in the cytoplasm A, suggesTo test whether ILF3 was required for AS Uchl1 nuclear entrapment, we ectopically expressed AS Uchl1 FL in ILF3-silenced HEK293/17 cells and checked its subcellular localization upon nucleocytoplasmic fractionation D. In conThe diversity of lncRNAs’ activity and function mainly depends on their modular architecture and their physical interactions with proteins. To understand the basic rules of this molecular network, we need to identify RNA sequences able to independently fold into functional secondary structures and the proteins that interact with them in a regulated fashion.in vitro phage display selection with NGS, this method provides an unbiased, high-throughput approach to study RNA-protein interactions. ORF phage libraries can faithfully represent whole proteomes or domainomes of cells, with the advantage of coupling phenotype to genotype identification. Furthermore, when ORF domain libraries are employed, the specific domains involved in bait-binding can be identified. Because this approach allows multiple screenings to be run on selected transcripts domains, we carried out 4 parallel selections, using 2 different RNA baits and 2 competitors to ensure reproducibility and robustness of our selection procedure. The RNA baits were 1) the invSINEB2 of AS Uchl1, where it exerts its ED function common to all mouse natural SINEUPs, and 2) AS Uchl1 Δ5′, an RNA lacking the BD but including the ED in an embedded format. This construct provides the backbone on which synthetic SINEUPs are built  and a dsRNA (By SPR analysis we measured invSINEB2-ILF3 binding kinetics  a dsRNA . It shou a dsRNA –65. The ILF3–AS Uchl1 interaction and its reliance on the embedded TE were then demonstrated by experiments in HEK 293T/17 cells upon ectopic expression of the lncRNA transcript. A reproducible enrichment of AS Uchl1 RNA was revealed in endogenous ILF3 immunoprecipitates, which was substantially reduced on deletion of invSINEB2. Although AS Uchl1 is a mouse transcript and ILF3 synthesized from the phage library and in HEK 293T/17 cells is of the human type, we considered these results also representative of the interaction in mouse given the 92% identity and 95% homology between human and mouse ILF3 protein sequences and the 100% conservation of dsRBM2, the invSINEB2 BD of ILF3. Future ILF3 immunoprecipitation experiments should be carried out in mouse cells to experimentally prove the interaction of the FL rodent protein with AS Uchl1. Nevertheless, because SINEB2 sequences are not present in the human transcriptome, we also demonstrated that ILF3 was able to bind FRAM, the ED in human natural SINEUPs. Although SINEB2 and FRAM do not present extensive homology at the primary sequence and there is no clear consensus sequence for ILF3 binding, our results suggest that they form conserved secondary structures that are able to bind common interacting partners. This result is relevant under the hypothesis that embedded TEs can act as convergent functional domains.We then asked whether ILF3-FRAM interaction is a representative example of a larger pattern of ILF3 binding to SINE sequences in the mammalian transcriptome. To this end we took advantage of ENCODE eCLIP data for ILF3 in 2 human cell lines. In general, ILF3 binding to SINEs was extremely strong, proving a highly significant and specific preference of ILF3 for transcribed fragments containing these elements. The presence of multiple additional ILF3 binding interactions with introns, coding exons, noncoding non-AS exons, and SINEs on the transcribed strand probably reflects the extensive functional diversity of the ILF3 gene in addition to an incomplete annotation of the transcriptome. It remains to be determined whether different levels of enrichment for Alu families reflect distinctive RNA secondary structures and protein binding profiles, opening up an interesting topic of investigation on the diversity of functional roles of embedded Alus in lncRNAs.in vitro and in vivo (Earlier data have demonstrated that TEs of the SINEs and Alu families are involved in the RNA association with nuclear protein complexes, which subsequently control RNA export and cytoplasmic availability , 67. Mor in vivo , we moniRecently, inverted repeat Alu elements embedded in long intergenic noncoding RNA-p21 have been shown to fold into specific structures required for RNA nuclear localization. Mutations disrupting such secondary structures resulted in altered long intergenic noncoding RNA-p21 distribution , 68. AccAs previously shown for other cellular systems –72, ILF3Therefore, the structural requirements for the embedded heterodimeric repeat composed of the invSINEB2 and the truncated Alu remain to be defined, along with the identity of additional protein partners and the details of their interactions with ILF3. In addition, future studies will investigate the biologic significance of a 20% increase in cytoplasmic AS Uchl1 RNA, including its effect on the ability to regulate endogenous protein levels of its RNA sense target.In summary, through the identification of ILF3 as a binding partner of mouse invSINEB2 and human FRAM embedded in SINEUP lncRNAs, we provide strong evidence that TEs act as functional modules in lncRNAs. By detailed bioinformatic analysis of eCLIP data, we showed that ILF3 binding sequences are highly enriched for SINEs embedded in human transcripts. We then demonstrated that nuclear localization of AS Uchl RNA depends on embedded TEs and is moderately influenced by ILF3. This work paves the way for further studies on the biologic role of interactions between ILF3 and embedded TEs in lncRNA dynamics and function.http://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails. Furthermore, a significant proportion of C2H2-ZNF mRNAs are retained in the nucleus. Intriguingly, both short poly(A) tails and nuclear retention can be specified by the repeated elements that encode zinc finger motifs. These Zinc finger Coding Regions (ZCRs) appear to restrict polyadenylation of nascent RNAs and at the same time impede their export. However, the polyadenylation process is not necessary for nuclear retention of ZNF mRNAs. We propose that inefficient polyadenylation and export may allow C2H2-ZNF mRNAs to moonlight as non-coding RNAs or to be stored for later use. The vast majority of C2H2-ZNF proteins have no unique biological function assigned to them; however, they are widely accepted as DNA binding proteins and those with an associated KRAB domain can recruit the transcriptional repressor KAP1/TRIM283. Recent studies have suggested that expansion of this family in primates occurred in part to combat the parallel expansion and evolution of transposable elements7. Notably, the human C2H2-ZNF genes have on average ~10 tandem copies of the ZNF motif2, which is sufficient to recognize a 30nt sequence, far longer than necessary for specificity8. Indeed, only ~60% of ZNF motifs were predicted to bind DNA in a recent analysis9. This gives rise to the possibility that some ZNF domains, or perhaps even the sequences encoding them, have other roles.Zinc-finger (ZNF)-containing proteins are among the largest families of proteins in the human genome. The Cys2His2 (C2H2)-type ZNF family has over 700 members, many of which are unique to primates and have arisen through gene duplication10 and the C2H2-ZNF motif has also been implicated in protein-protein interactions12. In addition, the linker domains between individual ZNFs are targeted by the TOPK/PBK kinase during mitosis to induce dissociation of these proteins from condensing chromatin14. Thus, these highly conserved repeated protein motifs may indeed serve functions other than binding DNA.In support of the zinc finger motif having additional functions, many C2H2-ZNFs were recently identified as putative RNA-binding proteins (RBPs)16. These miRNAs can simultaneously downregulate multiple C2H2-ZNF genes through translational repression and/or mRNA decay16. Second, C2H2-ZNF mRNAs are over-represented among transcripts with shorter than average poly(A) tails which could indicate unique processing or deadenylation mechanisms are at play17. Third, a large group of C2H2-ZNF transcripts are decayed more slowly in stem cells than in fully differentiated fibroblasts18. This could be facilitated by recognition of shared sequence elements by a stem cell or fibroblast-specific RBP or miRNA. Fourth, mRNAs encoding KRAB C2H2-ZNFs share similar RNA metabolism profiles including efficient synthesis and processing, and a tendency to be retained in the nucleus19. Next, the 3′ exons of C2H2-ZNF genes have unusual chromatin modification (H3me3K9) suggesting that they may auto-regulate through recruitment of the KAP1/TRIM28 transcriptional repressor21. Interestingly the level of H3me3K9 modification is strongly correlated with the number of ZNF motifs in the 3′ exon21. Finally, C2H2-ZNF genes are significantly over-represented among genes that have acquired Alu insertions22 which again may facilitate coordinated control either at the level of transcription or through ADAR-editing23.It is also possible that the DNA or RNA sequences encoding C2H2-ZNF domains have an additional, non-coding function. Sequence elements shared between different genes can facilitate coordinated control through recruitment of regulatory factors (such as RBPs and miRNAs). Several lines of evidence suggest that C2H2-ZNF genes share regulatory elements that allow their expression to be coordinated. First, four miRNAs can target the repeated regions encoding zinc fingers found in related C2H2-ZNF mRNAs17 and that they have an increased propensity to be retained in the nucleus19. In mammalian cells, a poly(A) tail of ~250 nt is added to the 3′ end of the majority of nascent mRNAs24 and influences downstream metabolism. Failure to polyadenylate a nascent transcript limits its ability to form a RiboNucleoprotein Complex (RNP) that is competent for export and leads to rapid decay25. Once a polyadenylated transcript has been exported, the poly(A) tail, in complex with PABPC1, potentiates translation by helping to recruit initiation factors. Over time, the poly(A) tail can be shortened by various deadenylases26. When the tail is shortened to such an extent that PABPC1 can no longer bind, rapid decay of the mRNA body generally ensues27. Thus at steady state, most mRNAs exist as a population with poly(A) tails varying in length from ~20 to ~250 nt29. Exceptions to this are the histone mRNAs30, transcripts with poly(A) limiting elements (PLEs)32 and mRNAs bearing cytoplasmic polyadenylation elements (CPEs)33 which can persist with short or no poly(A) tail. Here, we show that repetitive sequence elements encoding C2H2-ZNF motifs (Zinc finger Coding Regions or ZCRs) act as poly(A) limiting elements, resulting in a sub-population of mRNAs that have short poly(A) tails. In addition, we provide evidence that C2H2-ZNF mRNAs are retained in the nucleus through mechanisms that do not require polyadenylation.Based on the evidence outlined above, we hypothesized that sequence elements found within the ORF or 3′UTR of C2H2-ZNF mRNAs are responsible for aspects of their unique metabolism. We focused specifically on the observations made through global analyses that many C2H2-ZNF mRNAs have significant populations with shorter than average poly(A) tails34. In contrast, histone mRNAs and many non-coding RNAs  fail to bind to oligo(dT) because they lack a poly(A) tail. Yang et al.17 identified a group of mRNAs in human H9 embryonic stem cells and HeLa cells that are “bimorphic”, in that a significant portion of the population fails to bind oligo(dT). Among these are many C2H2-ZNF mRNAs.Most mRNAs can be effectively captured by hybridization of the poly(A) tail to oligo(dT)17 and most of which share similar metabolism profiles (“biotypes”) as classified by Mukherjee et al.19 who evaluated various properties of mRNAs including nuclear/cytoplasmic distribution, synthesis, processing, translation and decay rates , some of which were previously identified as bimorphicFirst, we assessed whether the selected transcripts exhibited bimorphic behavior with respect to binding oligo(dT). Total RNA isolated from HeLa cells was fractionated using oligo(dT) magnetic beads and the proportion of each mRNA in each fraction was quantified by qRT-PCR. Control transcripts with poly(A) tails of known length were spiked in and evaluated in the same way Fig. . As show35 to evaluate the poly(A) tail length of GAPDH, PPIA, ZNF12 and ZNF627 mRNAs in the oligo(dT) bound and flow-through fractions  these mRNAs exhibit strikingly little adenylation. The transcripts detected have very short, if any, poly(A) tails  could either reflect a heterogeneous mixture of mRNAs with long and short poly(A) tails, or there may be a homogenous population having a poly(A) tail of an intermediate length (~12–25 nt) that binds inefficiently to oligo(dT). In order to distinguish these possibilities we used a qualitative RT-PCR-based assayons Fig. . The bouons Fig. . This isils Fig. . This su39. Moreover, KRAB-domain ZNF mRNAs were enriched among mRNAs with a profile that includes increased nuclear retention40. We therefore wondered whether these ZNF transcripts with short poly(A) tails were also retained in the nucleus. We isolated nuclear and cytoplasmic RNA populations and quantified the level of ZNF and control RNAs in each fraction by qRT-PCR  tails we performed a two-step fractionation, in which we separated the nucleus and cytoplasm, isolated total RNA from each fraction and finally bound the RNA to oligo(dT) to separate polyadenylated RNAs. We quantified the amount of mRNA in each fraction by qRT-PCR as before. The results shown in Fig. In order to further characterize the mechanisms responsible for restricting the length of the poly(A) tail and retaining these transcripts in the nucleus, we created a reporter constructs in which the ORF and 3′UTR of the ZNF12 mRNA were fused to Renilla Luciferase with an upstream intron and expressed under a tetracycline responsive promoter Fig. . The samThe PPIA reporter was primarily polyadenylated and cytoplasmic, while the ZNF12 reporter largely failed to bind oligo(dT) and was present mainly in the nuclear fraction Fig. . We alsoWe next utilized RNA-FISH to assess the subcellular localization of each reporter. As expected, the PPIA reporter was primarily cytoplasmic with minimal nuclear accumulation. In contrast, the ZNF12 reporter was distributed throughout the cell with significant nuclear staining. This pattern is consistent with the distribution of the reporter and endogenous ZNF12 between nuclear and cytoplasmic fractions and supports that sequences in the ORF and/or 3′UTR of ZNF12 can influence the localization of the reporter transcript. Interestingly, most cells exhibited accumulation of the reporter RNA in multiple nuclear foci Fig. .In order to narrow down the region of the ZNF12 mRNA required for nuclear retention and poly(A) restriction, we cloned the ZNF12 ORF and 3′UTR separately into the RLuc reporter Fig. . Surpris41. By aligning the ORF and 3′UTR to each other, we were able to identify ZNF-related sequence elements at the 5′ end of the 3′UTR as well as additional inactivated ZNF-related motifs within the 5′ half of the ORF closer to the KRAB domain  and the third containing the remaining canonical ZNF motifs (canZNF). We also made a construct bearing just the region of the 3′UTR that contains sequences that may have encoded functional C2H2-ZNF motifs in the past (utrZNF). As predicted, the KRAB only reporter behaved much like the PPIA control; the transcript was polyadenylated and exported to the cytoplasm. In contrast, all the reporters bearing ZNF-like sequences  behaved more like the full length reporter in that these mRNAs were predominantly nuclear and did not efficiently bind oligo(dT). There was a clear positive correlation between the level of polyadenylation and the cytoplasmic accumulation of the ZNF12 reporter constructs  tail on ZNF mRNAs and their nuclear retention. There are short C-rich stretches in the Zinc Finger Coding (ZFC) regions that could possibly recruit hnRNP K  and the consequent reduction in miRNA levels should allow polyadenylation and export to occur. Despite strong evidence for expression of ZNF-targeting miRNAs in HeLa cells48  tail length of the ZNF mRNAs. This is consistent with the fact that the 3′UTR and utrZNF reporter mRNAs, which lack binding sites for ZNF-targeting miRNAs, still have short poly(A) tails and are retained in the nucleus  signal itself, as the majority of our constructs contain the SV40 late poly(A) signal. As the sequences coding for ZNF motifs are targeted by miRNAss48 Fig. , depletis48 Fig.  although51. 4sU-labeled nascent RNAs were conjugated to biotin and isolated by binding to streptavidin beads. The eluted RNA was then bound to oligo(dT) beads as before, and ZNF mRNAs were assessed in each fraction by qRT-PCR. As shown in Fig. To determine whether ZNF mRNAs with very short tails fail to get polyadenylated immediately following 3′ end cleavage, or instead get polyadenylated efficiently and then deadenylated at a later time, we investigated the length of tail on nascent mRNAs. HeLa cells were treated with 4-thiouridine (4sU) for 10 minutes and then total RNA was isolated52. Heterologous transcripts bearing the MALAT1 3′ end signal are neither adenylated nor retained in the nucleus53. When we replaced the 3′ end of the full length ZNF12 and PPIA reporters with the MALAT1 3′ end signal, the PPIA reporter transcript was still efficiently exported but the ZNF12 reporter mRNA was retained in the nucleus  tails. In addition, these mRNAs exhibit a nuclear distribution consistent with failure to export, or with very rapid decay upon entry to the cytoplasm. There is a clear correlation between the level of polyadenylation and the efficiency of export for C2H2-ZNF mRNAs and the same sequence elements seem to influence both phenomena. Notably, both failure to polyadenylate and nuclear retention are conferred on a heterologous RNA by sequences capable of encoding C2H2 zinc finger motifs, which we have called Zinc finger Coding Regions or ZCRs. Furthermore, ZNF12 reporter mRNAs that do not experience polyadenylation are also retained in the nucleus supporting that nuclear retention can be separated from the polyadenylation process.54. Once the poly(A) tail becomes long enough to bind the nuclear poly(A) binding protein (PABPN1), an interaction between PAP, CPSF and PABPN1 causes the polyadenylation reaction to become more processive and the tail is rapidly extended to ~250 nt24. The C2H2-ZNF mRNAs appear to undergo cleavage, but the poly(A) tail is not extended beyond a few nucleotides suggesting that recruitment or activity of PAP may be impaired by factors associating with the ZCR. Alternatively, ZCR-binding factors could prevent recruitment of PABPN1 or other factors connected with poly(A) tail length control such as ZC3H1455.Polyadenylation initiates with recognition of the poly(A) signal (AAUAAA) upstream of the cleavage site and a downstream element (DSE) by Cleavage Polyadenylation Specificity Factor (CPSF) and Cleavage Stimulation Factor (CstF) respectively. Following cleavage, poly(A) polymerase (PAP) begins to add adenosine residues to the 3′ end of the mRNA57. However, this phenomenon requires binding of the inhibitory factor relatively close to the poly(A) signal while ZCRs are able to function from a distance. In addition, inhibition of polyadenylation by splicing factors generally results in reduced mRNA abundance suggesting that the unadenylated transcripts are degraded but the C2H2-ZNF mRNAs are clearly able to persist despite their short poly(A) tails. ZCRs bear more functional similarity to poly(A) limiting elements (PLE) which can be located in the coding region, within the last exon, and can restrict polyadenylation even in the presence of a heterologous strong poly(A) signal31. However, unlike the C2H2-ZNF mRNAs, PLE-containing mRNAs appear to be exported and translated efficiently despite lacking a poly(A) tail32. The mechanism by which PLEs limit polyadenylation is not understood, although the splicing factor U2AF65 associates with PLEs and can modulate their activity58. Notably, when we prevented polyadenylation, by replacing the poly(A) signal with the MALAT1 3′ end sequence, the ZNF12 reporter mRNA was still retained in the nucleus suggesting that any trans-acting factors involved in nuclear retention are not recruited by the polyadenylation machinery.One characterized mechanism to inhibit polyadenylation is through binding of splicing factors that can inhibit PAP close to the poly(A) site43 or the AGCCC element in BORG59) is sufficient to prevent export by recruiting a protein factor such as hnRNP K. Another protein able to block RNA export is the ZFC3H1 exosome adaptor which can compete with the export factor ALYREF for binding to the RNA60. In the case of lncRNAs like MALAT1 and NEAT1, the minimal region required for nuclear retention is much longer (100 s of nucleotides) and may function by providing structural scaffold for proteins to bind and/or regions for RNA-RNA hybridization61. It appears specific properties of these lncRNAs allow them to nucleate phase separation and accumulate in subnuclear compartments62. Given that the ZNF12 reporter accumulates in foci  tail, but this is not sufficient on its own to result in nuclear retention: The MALAT1 3′ end cannot specify nuclear retention of the PPIA reporter  signal, although it is not clear whether this transcript actually acquired a poly(A) tail61. Based on this result we propose that the ZFCs function through a pathway similar to that utilized by MALAT1. In this respect, we note many non-coding RNAs that undergo canonical splicing and polyadenylation, including the Xist66, Bsr67 and BORG59 lncRNAs, are retained in the nucleus through various mechanisms, and in a variety of subnuclear locations68. Certain factors binding the ZCR could perhaps reduce recruitment of export factors by partitioning these mRNAs to inaccessible nuclear domains or by preventing interactions between the export machinery and mRNA processing factors.Nuclear export requires assembly of an export-competent RNP which generally depends on accurate and complete processing of the transcriptter Fig.  or of a Alu elements are retained in the nucleus, in paraspeckles, due to extensive post-transcriptional A-to-I editing69. Alu elements also contain C-rich sequences that drive nuclear localization through binding of hnRNP K43. Although C2H2-ZNF genes are more likely to contain Alu-derived sequences than other transcripts23, and C2H2-ZNF mRNAs are significantly enriched among a group of 333 genes with 3′UTR IR-Alu elements70, this mechanism cannot explain our observations for several reasons. First, only one of the six ZNF mRNAs we studied here (ZNF43) retains IR-Alu sequences in the mature transcript and experiences editing70. Second, unlike the C2H2-ZNF mRNAs, the vast majority of Alu-containing mRNAs are polyadenylated17. Third, there is no evidence for association of hnRNP K with the transcripts we evaluated46 and hnRNP K knockdown had no effect on localization of the ZNF mRNAs  NAs Fig. . Finallylls Fig.  and C2H2Alu repeats that are extensively edited, leading to nuclear retention as described above. However, in response to stress, the CTN transcript is processed to remove the edited region and generate the mature mCAT2 mRNA72. The mCAT2 mRNA is efficiently exported and translationally competent. A more widespread pathway for delaying nuclear export is through retention of introns74. Transcripts with one or more unspliced introns can remain in the nucleus until an appropriate stimulus induces completion of splicing and allows formation of an export competent RNP75. Although our reporter transcripts have an intron and undergo splicing, the ZFC element can still retain RNAs within the nucleus. Further investigation is required to determine whether a poly(A) tail or the polyadenylation experience, can overcome this retention. If this were the case, then polyadenylation could perhaps induce rapid export and translation when needed, such as during certain phases of the cell cycle, or in response to stress.The retention of C2H2-ZNF mRNAs within the nucleus presumably prevents them from functioning as mRNAs. Nuclear retention is an established mechanism to regulate gene expression. For example, the CTN non-coding RNA contains IR-76. One particularly relevant example is the Signal Sequence Coding Region (SSCR) found at the 5′ end of transcripts encoding secretory proteins which both encodes signal peptide and enhances RNA export77. In this case, the SSCRs role as an mRNA export element does not compete with its role in coding for protein because the two functions are required sequentially. However, in other examples a non-coding function can compete with the coding function of an mRNA. This is the case for the bifunctional Steroid Receptor RNA Activator, SRA, which acts as an RNA scaffold to assemble factors involved in nuclear receptor signaling78. An alternatively spliced isoform of the SRA RNA encodes a protein that plays a role in trans-activation of nuclear steroid hormone receptors in the nucleus. The coding isoform is exported to the cytoplasm and translated and thus cannot act as a nuclear scaffold while the non-coding isoform lacks appropriate start codons, fails to be exported and cannot be translated to make protein79. It seems possible that the C2H2-ZNF mRNAs have acquired a nuclear non-coding function such as acting as a scaffold or miRNA decoy.There are several instances where coding region sequence elements moonlight to regulate mRNA export or other aspects of mRNA metabolismtrans-acting factors that associate with the ZCRs to prevent polyadenylation and/or export. We are also interested in characterizing the nuclear domain these transcripts are accumulating in and evaluating whether cellular conditions such as stress or the cell cycle influence export and/or polyadenylation of these RNAs. Such studies might eventually allow us to modulate the polyadenylation/export of the entire class of C2H2-ZNF bimorphic transcripts and discern the biological impact.Future experiments will aim at characterizing the ZCRs in more detail, and identifying in vitro transcription of control RNAs with different lengths of poly(A) tail, fragments of the S. cerevisiae ACT1, MET3 and MET25 genes were amplified by RT-PCR using primers shown in Table BamHI and SalI sites of pGEM4 (Promega) plasmids that had adenosine stretches of 15, 45 or 149 adenosines, respectively, inserted 3′ of the multiple cloning site. These plasmids were linearized with PvuII and the empty pGEM4 plasmid was linearized with SmaI (to generate an RNA with no poly(A) tail). Each linearized plasmid was in vitro transcribed using SP6 RNA polymerase (Thermo Fisher Scientific) and the resulting RNA was gel purified before use.In order to generate templates for ApaI site of pTRE3G-BI-ZsGreen (Clontech), and then inserting Renilla luciferase amplified from pLightSwitch (Switchgear Genomics) into the BglII/NotI sites. The empty parent vector was digested with Not1. Fragments containing ORF and/or 3′UTR sequences from ZNF12 (NM_016265.3) and PPIA (NM_021130.4) were amplified from HeLa cDNA or from previously generated plasmids. The 3′ end of the mouse MALAT1 gene (NR_002847.3) was synthesized as a gBlock (Integrated DNA Technologies) and used to replace the 3′ end of PPIA or ZNF12 in the original clones. Individual constructs were created using In-Fusion HD (Clontech) or NEBuilder HiFi (New England Biolabs) which are ligation independent cloning kits. Primers for the PCR reactions are shown in Table E. coli. All plasmids were sequenced prior to use.The parent vector for ZNF12 and PPIA expression constructs was generated by cloning the β-globin/IgG chimeric intron from pCI-Neo (Promega) into the 2. HeLa Tet-Off Advanced cells (Clontech) were maintained in DMEM supplemented with 10% Tetracycline-free FBS (Clontech), 2 mM L-glutamine, 0.37% sodium bicarbonate, and 100 μg/mL G418 to maintain expression of the transactivator.HeLa S3 cells adapted to adherent culture were maintained in DMEM (4.5 g/L glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 0.37% sodium bicarbonate at 37 °C in 5% CO2.Induced Pluripotent Stem (iPS) cells  were cultured on Matrigel (BD Biosciences) in mTesR1 media (STEMCELL Technologies) at 37 °C in 5% CO2. Human foreskin fibroblasts  were maintained in DMEM (4.5 g/L glucose) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 10% FBS at 37 °C, 5% COHeLa Tet-Off Advanced cells were transfected with reporter plasmids using jetPRIME (Polyplus) prior to allowing them to adhere to the dish. A ratio of 3 µL reagent to 1 µg of plasmid was employed according to the manufacturer’s recommendations. Cells were harvested 24–48 hr after transfection. Transfections were evaluated for ZsGreen expression using a Nikon Diaphot 200 microscope with a GFP-B filter cube (Nikon). Transfection efficiency was generally >70% at ~48 hours post transfection.80. Cells were scraped into ice cold phosphate buffered saline (PBS), collected by centrifugation, resuspended in NP40 lysis buffer  and incubated on ice for 5 min. Nuclei were pelleted by centrifugation for 5 min at 500 × g at 4 °C, the supernatant/cytoplasm was transferred to a fresh tube and an equal volume of TRIzol was added. After washing with NP40 lysis buffer, the nuclear pellets were lysed in TRIzol. RNA was isolated as described below. The RNA from the nucleus and cytoplasm was resuspended in an equal volume such that an equal number of cells were represented in 1 µl regardless of RNA concentration.Nuclei and cytoplasm were separated as described previouslyDpnI restriction enzyme if plasmid DNA had been transfected into the cells. DNase was removed by phenol/chloroform/IAA extraction and ethanol precipitation.TRIzol was added to cells after removal of the media. RNA isolation was performed according to the manufacturer’s instructions except an additional phenol/chloroform/isoamyl alcohol (25:24:1) extraction was performed immediately prior to addition of isopropanol. Total RNA was treated with DNAse I (Thermo Fisher Scientific) or with TURBO DNAse (Thermo Fisher Scientific) and 81.Reverse transcription was performed in 20 µL reactions with Improm-II Reverse Transcriptase (Promega) using 0.5 µg of random hexamers, according to the manufacturer’s instructions. 1–2.5 µL of cDNA was used to set up triplicate qPCR reactions using IQ SYBR-Green Supermix (BioRad) and primers as listed in Supplementary Table in vitro transcribed control RNAs with poly(A) tails of 0, 15, 45 and 149 nt were spiked into 10–20 µg total RNA samples in binding buffer  prior to fractionation. Oligo(dT)25 magnetic beads (New England Biolabs) were equilibrated in binding buffer. RNA samples were denatured at 70 °C for 2 min before being mixed with the beads and incubated at room temperature for 10 min. A magnetic field was used to retrieve the beads and the unbound fraction was reserved. The beads were washed with wash buffer  containing 1 µL Ribolock RNase Inhibitor (Thermo Fisher Scientific) and the wash was added to the unbound fraction. This step was repeated with wash buffer and again with low salt buffer , with the flow through being added to the unbound fraction each time. Finally, the poly(A)+ RNA was eluted in Tris-HCl, pH 7.5, 1 mM EDTA at 50 °C for 2 min. This step was repeated. The RNA in each fraction was recovered by precipitation with glycogen carrier and resuspended in 20 µL of nuclease-free water. Equivalent volumes of the bound (poly(A)+) and unbound(poly(A)−) fractions were used to make cDNA.80 fmol of a mixture of 50.HeLa cells were treated with 500 µM 4-thiouridine for 10 min and then scraped into TRIzol. RNA was isolated using the RNAeasy kit (Qiagen) according to the manufacturer’s protocol. 75 µg of total RNA was labeled with biotin and fractionated using streptavidin magnetic beads (Miltenyi Biotec) as described previously35. Briefly, a population with no poly(A) tail was generated by treatment with oligo(dT)18 and 5 U of RNAse H  for 30 minutes. Next, 1 µg of this treated sample and an untreated RNA sample were each ligated to a linker RNA  using T4 RNA Ligase 1 (New England Biolabs) at 16 °C overnight. The RNA was recovered and converted to cDNA using a primer complementary to the linker (5′-CTGGAATTCGCGGTT-3′) and Improm II Reverse Transcriptase (Promega). Finally, the 3′ end of each transcript of interest was amplified by PCR using OneTaq Hot Start Polymerase (New England Biolabs) employing a gene-specific forward primer paired with the linker primer as the reverse primer. The annealing temperature (55–59 °C) and number of cycles (27–32) were adjusted to optimize the amount of product for easy visualization. PCR products were separated and visualized by agarose gel electrophoresis and imaged on a BioRad Gel Doc EZ Imager. Image Lab Software (BioRad) was used to determine poly(A) tail lengths.The LLM-PAT assay was performed as described previously32P-end-labeled DNA oligonucleotides (resuspended in pre-hybridization buffer) specific to the gene of interest (Actin [5′-CATAATTTACACGAAAGCAATGCTATC-3′] or ZNF12 [5′-CACAAATATGCTATGAATATAG-3′]) overnight. The following day, blots were washed (6xSSC/0.1%SDS) three times at room temperature for 5 minutes followed by an additional wash at 50 °C for 20 minutes. Following an overnight exposure, phosphor screens were scanned using a Typhoon Trio  and the results analyzed with ImageQuant software .HeLa Tet-Off Advanced cells were transfected with the ZNF12 reporter plasmid as described above. Cells were then collected in TRIzol and total RNA was prepared. 10 µg of total RNA was hybridized to a gene-specific DNA oligonucleotide (Actin [5′-GTGGACTTGGGAGAGGACTG-3′] or ZNF12 [5′-TATACAACTTACCAGATGTAATT-3′]) complementary to a region just upstream of the poly(A) signal. Oligo(dT) was included as indicated. Following treatment with 5 U of RNAse H  for 30 minutes, RNA was recovered and separated on a 5% denaturing polyacrylamide gel. RNA was then transferred to a nylon membrane  followed by UV crosslinking. The blots were then pre-washed (0.1xSSC/0.1%SDS) at 65 °C for 1 hour, pre-hybridized  at 42 °C for 1 hour and then probed using 82. Pools of stably transduced cells were selected with 10 μg/mL puromycin and then maintained in 1 μg/mL puromycin.For DICER knockdown, HeLa S3 cells were transduced with lentiviral particles containing DNA encoding an shRNA that targets DICER (Sigma-Aldrich:TRCN0000051261) or a negative control lentiviral particles generated using the pLKO-1 empty vectorTo transiently knockdown HNRNPK, HeLa Tet-Off Advanced cells were transfected with a pre-designed siRNA targeting HNRNPK  or control siRNA (Sigma SIC001) at a final concentration of 50 nM using jetPRIME (Polyplus) reagent in accordance with the manufacturer’s protocol. Knockdown was assessed 48 hours after transfection.In each case, knockdown was verified by qRT-PCR using primers described in Table HeLa Tet-Off Advanced cells were transfected with PPIA or ZNF12 reporters and plated on coverslips. After 24–30 hr, cells were fixed and permeabilized using 3:1 methanol:acetic acid for 10 min at room temperature and an additional 2 hr at 4 °C. After washing in Wash Buffer (10% deionized formamide in 2xSSC) the cells were treated with Stellaris RNA-FISH hybridization buffer containing 125 nM of Stellaris RNA-FISH Q570 probe targeting Renilla luciferase  at 37 °C overnight. Coverslips were washed in Wash Buffer and then in 1xSSC before mounting with Prolong Diamond AntiFade mountant with DAPI (Thermo Fisher Scientific). The cells were visualized using an Olympus IX71 inverted fluorescent microscope at 100X magnification using the 31000 DAPI/Hoechst filter  to visualize DAPI and the 41002 TRITC (Rhodamine)/Cy3 filter  to visualize Quasar570. Images were captured using Q Imaging Retiga 2000R camera.To determine the percentage of an mRNA bound to oligo(dT) the amount of each mRNA in equal volumes of the bound and flow through fraction was assessed by qRT-PCR and added to give the total. For graphing and subsequent analysis the amount bound to oligo(dT) was expressed as a percentage of this total. Similarly, to determine the percentage of an mRNA in the cytoplasm the amount of each mRNA in equal volumes of the cytoplasmic and nuclear fractions was assessed by qRT-PCR and added to give the total. The amount in the cytoplasm was expressed as a percentage of the total for graphing and analysis.83 was used to demonstrate equal variance, then a one-way ANOVA test was performed with post-hoc Tukey test84 or Dunnett’s test as indicated. P values of less than 0.05 were considered significant.Three independent replicates were performed for each experiment unless otherwise noted. For pairwise comparisons, a paired two-tailed t-test was employed. For multiple comparisons, Levene’s test85.Much of the work described was published in a thesis by Aimee L. JalkanenSupplemental Figures