We report a systematic investigation of the temperature dependence of electrical resistance behaviours in tri- and four-layer graphene interconnects. Nonlinear current–voltage characteristics were observed at different temperatures, which are attributed to the heating effect. With the resistance curve derivative analysis method, our experimental results suggest that Coulomb interactions play an essential role in our devices. The room temperature measurements further indicate that the graphene layers exhibit the characteristics of semiconductors mainly due to the Coulomb scattering effects. By combining the Coulomb and short-range scattering theory, we derive an analytical model to explain the temperature dependence of the resistance, which agrees well with the experimental results. The e2 V−1 s−1-12. Most2 V−1 s−1,14 and t2 V−1 s−1. As more-1/2 dependence shows the evidence of the electron–electron Coulomb interaction in FLG. Our temperature-dependent resistance results reveal that the FLG interconnects display semiconductor properties and further confirm that Coulomb interaction can play a dominant role.In this letter, we report a systematic investigation of the temperature dependence behaviour of the four-terminal electrical resistance in FLG interconnects. The resistance of tri- and four-layer graphene, under direct current (DC) electric fields and in a temperature range from 5 to 340 K was measured. The T2 substrate. The number of graphene layers was confirmed by micro-Raman spectroscopy through the 2D-band deconvolution procedure [−7 mbar. Electronic transport measurements were performed on multiple samples, using the physical property measurement system  with a fixed excitation current of 0.01 mA; the temperature varied from 5 to 340 K.The graphene layers were produced by mechanical exfoliation techniques  from bulrocedure . The Ramrocedure ,17. ShowI-V) characteristics at different temperatures of (a) tri- and (b) four-layer graphene interconnects. Insets show the enlargement of the measurement results at low electric fields. For the tri- and four-layer graphene, the interconnect resistors display two distinct regions of ohmic characteristic: one at fields larger than 0.01 V/μm but less than 0.10 V/μm and the other at fields larger than 0.10 V/μm. The nonlinear behaviour of current–voltage characteristics at low threshold (<0.10 V/μm), and the second ohmic region in the strong DC electric field (>0.10 V/μm) can be explained by the heating effect [I-V over the whole temperature measurement range.Figure g effect . Within g effect ,19. At sGd = dI/dV of (a) tri- and (b) four-layer graphene as function of the temperature T−1/2 on a semi-logarithmic scale. As shown in the Figure In order to study the existence of electron–electron Coulomb interaction and how it plays an important role in our system, we adopted the resistance curve derivative analysis (RCDA) method to investigate the dominant scattering mechanism . Figure E. Shown in Figure RT/RT = 5K as a function of the electric field E at different temperatures, for (a) tri- and (b) four-layer graphene interconnects. Insets display the optical micrographs of the FLG interconnect. At a lower temperature range of 5 to 50 K, as the electric field increases from 0 to 0.6 V/μm, the resistance of the tri- and four-layer graphene interconnects show a reduction of about 30% and 70%, respectively. However, for the temperature range T ≥ 200 K, the corresponding resistance drop is smaller. The larger drops of the resistance at lower temperature range indicate that Coulomb scattering is the main transport mechanism in the FLG interconnects at this temperature range as it is proportional to the carrier density. Hence, Coulomb scattering is strongly dependent on temperature. We further note that with increasing temperature, the observed results indicate that the scattering induced by electric field from the substrate surface polar phonons is significantly screened by the additional graphene layers at room temperature [There are many experimental and theoretical reports on the mobility of one- to three-layer graphene from two- and four-terminal measurements. The main scattering mechanisms in one- to three-layer graphene are Coulomb scattering -23, shorperature ,22.RT/RT = 5K, whereby we investigate the temperature dependence change of the resistance. In Figure RT/RT = 5K is observed for decreasing temperature for both the tri- and four-layer graphene. This R-T behaviour indicates that the carriers transport in the graphene layers is non-metallic in nature. This implies that, the resistance does not originate from thermal activation but is attributed to ES VRH between localized states induced by the charge impurities [In order to further study the VRH and localized insulating behaviour, we investigate temperature dependence of electronic transport measurements on a tri- and four-layer graphene. Figure purities -23. ThisAt low temperature, the main scattering mechanisms in graphene are largely due to the Coulomb impurity and the short-range defect scatterings . Based o2-graphene interface, and the short-range scattering is constant. Then the energy average scattering time is deduced as [Based on a model proposed by Hwang et al. , we can duced as ,22:Ek is the wave vector energy and τ(Ek) is the transport scattering rate. For the low temperature limit, the scattering time averaged over energy can be written as 〈τ〉 ≈ τ(EF) [D(EF) in tri- and four-layer graphene is assumed to be a constant where  ≈ τ(EF) . The denructures -23.3-4 layers ∝ kBT) [In the high temperature regime, the Coulomb scattering is a strong function of temperature while the short-range scattering is independent of temperature. This is attributed to the density of states, the matrix element of graphene and the screening function being energy independent in FLG -23. Hencs ∝ kBT) . For triRsr−3–4 layers = C, and A, B and C are the fitting parameters. In our measurements, we have observed a linear approximation for the temperature-dependent normalized resistance of tri- and four-layer graphene:where we have defined These considerations explain qualitatively why the resistance of tri- and four-layer graphene decreases with the increasing temperature. We note that due to the complexity of the FLG band structure, these anomalous electrical properties are believed to originate in the unusual band structures near the Fermi level of graphene -29. MoreI-V curve shows unique combination of the low threshold of linearity and manifestation of the second ohmic region for the strong DC electric field in the FLG interconnects. With the RCDA method, our experimental results suggest that Coulomb interaction plays an essential role. The non-metallic temperature-dependent resistance is observed in the temperature range of 5 to 340 K. In this case, even though the FLG band structure as semimetal with zero-band gap, tri- and four-layer graphene resistors behave more like semiconductors. By combining the Coulomb and short-range scattering theories, an analytical model was developed, which well explains the experimental results.In conclusion, FLG interconnects were fabricated via a combination of mechanical exfoliation and photo lithography techniques. The temperature dependence of the electrical resistance of tri- and four-layer graphene was investigated. The observed The authors declare that they have no competing interests.YPL fabricated the device and performed the experiments. WQJ and WSL coordinated the project. ZWL and WSL provided key interpretation of the data. QJW and WSL drafted the paper. All authors read and approved the final manuscript.


Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.Uncommonly large size of spliceosomal intron was observed in In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome. Eukaryotic genes contain spliceosomal introns that are post-transcriptionally removed by the spliceosome, an RNA-protein complex . The lenPlant genomes usually have compact genes due to small introns . ArabidoTransposable elements (TEs) are known to be major components of plant genomes . The oriTo further investigate intron size evolution in plant genomes, we present a novel approach to identify and analyze introns of large size using genomic data for a domesticated grapevine cultivar (Vitis vinifera Pinot Noir). The specific objectives are to investigate the size distribution of introns; relationship between intron size and gene function, determine the contents of introns and examine the associations between intron size evolution and grapevine domestication. Generally, we show that introns of truly extraordinary size are widespread in cultivated grapevines, a phenomenon not observed in other plant genomes.Arabidopsis proteins and 15,683 proteins show homology to Populus proteins . BLAST searches against an EST database of other Vitis species showed that 15,089 clusters (60.5%) had high similarity hits in at least one other species of grapevine.In total, 353,748 publicly available EST sequences of V. vinifera were clustered into 26,111 clusters, 8,560 of which represented by only one EST sequence. The largest cluster is composed of 1,998 EST sequences. The distribution of cluster size suggested that one-third of V. vinifera genes are not expressed at very high levels (32.8% singletons) and a few (3 clusters contain more than 1000 ESTs) genes are highly expressed across the representative EST libraries. Among the 26,111 clusters, 5,678 clusters contain no intron, while 20,433 clusters were predicted to be spliced. Among all clusters, 17,059 clusters were predicted to encode proteins, in which 15,369 predicted proteins show homology to Vitis vinifera Pinot Noir, is slightly larger than other sequenced plants [The average intron size, predicted by the annotation of the first draft sequences of d plants . After mComparisons between the 2563 clusters (predicted genes from EST clusters) and existing gene annotations of the V. vinifera genome revealed that 1,812 clusters matched to known annotations with 100% identity, with 1,137 matches that showing identical transcription start/end sites and exon/intron structure between our predictions and known annotations. However, in 217 matches, multiple EST-predicted genes (487 clusters) matched to single annotated genes, while 188 clusters matched to multiple annotated genes. The later case represented one type of annotation error in V. vinifera genome, in which a single gene supported by expression data was incorrectly split into multiple annotated genes due to a large intron between coding regions. Among the 188 clusters, 99 clusters correspond to multiple, consecutively annotated genes, suggesting that additional introns are needed between annotated genes to correct the annotations  were identified , suggesting younger LTR retrotransposon insertions and recent LTR retrotransposition activity in these introns. Based on an estimate of substitution rate in LTR regions, age estimates of LTR retrotransposons in introns identified a burst of LTR retrotransposon activities: <10,000 ya (LTR similarity 100%)  Figure . For theIn the 39 genes with identical exon/intron structure/number in Arabidopsis and Populus, repetitive elements comprised roughly 90% of the 74 intronic regions, in which 10 complete LTR retrotransposons were identified. Among the 74 introns, 1 intron contains only a LINE; 1 intron contains only LTR elements; 15 introns contain LINEs and other repetitive elements; 15 introns contain LTR elements and other repetitive elements; 5 introns contain LTR, LINEs and other repetitive elements; 34 introns contain only other repetitive elements; and 3 introns do not contain repetitive elements. According to a BLAST search against the V. vinifera genome, 'other repetitive elements' were highly repetitive within V. vinifera and likely represented un-classified or unnamed grapevine-specific TEs. The three introns without any identified repetitive elements all contain LINE-like ORFs otherwise highly repetitive in the V. vinifera genome.Characterizations of four selected introns  in Vitis species and domesticated varieties revealed intron length variation across these species and varieties Table . None ofGenome-wide intron analysis has only been done in very few plants . In thisAnnotation errors such as splitting single gene into multiple ones as a result of unsually long introns, were revealed by comparing ESTs to current annotations Figure . This waLarge size introns are expected to require more energy to be transcribed and spliced, so large introns may decrease gene expression (energy cost hypothesis) . Such hyIn contrast, gene size and/or intron size seems to be not related to gene function in this study. Among identified large genes, there is no particularly overrepresented gene family. There are still about 20% of expanded intronic regions not accounted for by similarity searches or repeat masking, suggesting unique sequences in introns are also evolving. Most of these unique sequences may be evolving neutrally since there may not be any selective constraint on them. However, a few of them may contain very important regulatory elements controlling gene expression patterns thus under selective constraints.Populus specific intron contraction can explain the intron size difference between the two plants. The young LTR retrotransposons and unique presence of these TEs in domesticated grapevine both suggested that an intron size expansion is more likely to be the case. Wendel et al. [Comparisons of genome sizes and intron sizes between Populus and Vitis revealed that intron sizes has increased much more than genome size. Either grapevine specific intron expansion or l et al. ,24 propoA major force behind the intron size expansion in grapevine is the proliferation of TEs, consistent with previous observations . Among vV. vinifera expanded introns contain significantly more identical LTR pairs, suggesting that more young LTR retrotransposons have been preserved in introns of domesticated grapevine  were obtained from NCBI GenBank using the PartiGene package . Only V.artiGene ) and TGIartiGene . Both meartiGene  searchesartiGene . The proartiGene .EST consensus sequences were mapped to genomic sequences of V. vinifera Pinot Noir  to identV. vinifera proteins were subjected to HMM search of protein functional domains against Pfam database [Genes with putative large introns were submitted to the following analysis: 1, the coding regions were predicted and translated into protein sequences by ESTScan ; 2, preddatabase .Identified large introns (3 kb~100 kb-long) were subjected to the following analysis: 1, the intronic sequences were screened by RepeatMasker  to identTo characterize homologous introns in other Vitis species and varieties, intronic regions that met the following criteria were chosen: 1, there is only one repetitive element in the intron, suggesting a relatively recent expansion; 2, the total intron size without repetitive elements does not exceed 3 kb (to facilitate PCR amplifications). Primers were designed for the flanking exon sequence as well as conserved regions within the repetitive elements to allow scoring either the presence or absence of the elements in a given intron (primer sequences available upon request). PCR amplifications of selected intronic regions were performed for one individual each of six wild grapevine species: V. rotundifolia, V. californica, V. girdiana, V. aestivalis and V. labrusca, V. jacquemontii, and seven V. vinifera cultivars: Cabernert Sauvignon, Chardonnay, Dolcetto, Pinot Noir, Riesling, Sangiovese and Zinfandel. Alleles with or without TE insertions were determined by the size of the PCR product.The authors declare that they have no competing interests.KJ and LRG conceived the study, designed and carried out the experiments, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.


However, it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3′ overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3′ end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate  Jumping genes are DNA sequences present in the genome of most living organisms. They contribute to genome dynamics and occasionally result in hereditary genetic diseases or cancer. L1 elements are the only autonomously active jumping genes in the human genome. They replicate through an RNA–mediated copy-and-paste mechanism by cleaving the host genome and then using this new DNA end as a primer to reverse transcribe its own RNA, generating a new L1 DNA copy. The molecular determinants that influence L1 target site choice are not fully understood. Here we present a quantitative assay to measure the influence of DNA target site sequence and structure on the reverse transcription step. By testing more than 65 potential DNA primers, we observe that not all sites are equally extended by the L1 machinery, and we define the rules guiding this process. In particular, we highlight the importance of partial sequence complementarity between the target site and the L1 RNA extremity, but also the high level of flexibility of this process, since detrimental terminal mismatches can be compensated by an increasing number of interacting nucleotides. We propose that this mechanism contributes to the distribution of new L1 insertions within the human genome. Retrotransposons are highly repetitive and dispersed sequences, accounting for almost half of our DNA cis with their encoding mRNA L1 retrotransposition starts with the transcription of a 6 kb L1 RNA driven by an internal Pol-II promoter The current model for non-LTR retrotransposon integration, named target-primed reverse transcription (TPRT), was originally deduced from biochemical studies on the insect R2Bm element 5′-TTTT/A-3′) and are often preceded by T-tracts of variable length in vitro, without apparent sequence or structure requirement, or any need for primer-template complementarity As regards L1, conclusive evidence on whether primer-template complementarities are required for efficient reverse transcription initiation is lacking. Most L1 pre-integration sites contain an EN recognition sequence  tail of the L1 RNA for efficient priming of reverse transcription. To achieve this goal, we validated a direct L1 extension assay (DLEA) to quantitatively measure the ability of native L1 RNPs to initiate reverse transcription. Then we systematically assayed more than 65 DNA substrates varying in sequence and structure, allowing us to define the preferential rules of L1 reverse transcription priming. Our results clarify the importance of base-pairing between the L1 RNA template and the target site DNA for this process and demonstrate its exceptional flexibility.To test the DNA primer requirements for initiating L1 reverse transcription, we set up a direct L1 extension assay (DLEA), which would avoid PCR and therefore would allow us to quantitate L1 priming efficiencies. The L1 retrotransposition machinery is notoriously difficult to express and to detect in most experimental systems. To obtain sufficient amounts of L1 RNPs for direct detection, we modified the protocol developed by Kulpa and Moran 18 primer in the presence of radiolabelled 32P-dTTP. This reaction would mimic the initiation step of L1 reverse transcription, which starts at the poly(A) tail of the L1 RNA. After a 4 min incubation at 37°C, we purified the reaction products and resolved them on sequencing gels. A short end-labeled oligonucleotide was added after the reaction as a recovery control (RC). No or minimal extension was detected in vector or RT-defective controls consistent with the presence of only minimal amounts of endogenous hL1 activity in RNP preparations . We chose to use 2 µg of RNPs, at the upper end of the linear range, for all following experiments and to set to 100% the level of extension obtained with an oligo(dT)18 primer under these conditions. Based on the dynamic range of the initial RNP titration, primer extension efficiencies as low as 5% should therefore be reliably quantified. The products are heterogeneous in length, consistent with the expected products of poly(A) reverse transcription and range from 19 nucleotides (nt) to approximately 150 nt (We reasoned that if L1 RNPs were active enough we should detect the extension of an oligo(dT)arations . In contarations , showingin vivoTo further confirm that the ladder observed results directly from the reverse transcriptase activity of the transfected L1 element, we performed additional controls. RNase treatment reduced primer extension to undetectable levels , the reabona fide initiation of reverse transcription by native mammalian L1 RNPs through the direct incorporation of radiolabeled dTMP in a primer extension reaction. Importantly, DLEA is quantitative since it demonstrates a linear relationship between the signal and RNP quantities under the reaction conditions employed.Altogether these results show that DLEA detects 18 primer to a non-T nucleotide in order to prevent base-pairing of the primer 3′ end to the L1 poly(A) tail , in agreement with previous reports 3 to V6), DNA polymerization becomes undetectable under the employed reaction conditions  tail . Althougnditions  as a prinditions , lane 8.Next, we measured the influence of each individual terminal base on primer extension. Although all terminal mismatches reduced the efficiency of reverse transcription initiation to some extent, a terminal G was the most detrimental, whereas a C or an A was better tolerated . Thus th1 to T6). Initiation of reverse transcription is robustly detected only when the single-stranded primer ends with at least 4 Ts and trace activity can already be detected with 3 terminal Ts  RT to prime reverse transcription under identical experimental conditions. In these experiments, exogenous poly(rA) was added as a template together with quantities of the AMV RT that lead to similar levels of extension as the L1 RNP using the (dT)et al. observed processing in 33/81 (39%) of the analyzed clones obtained with HeLa cells, while Kopera et al. found 5/45 (11%) of processed primers in CHO-derived cell lines. In comparison, we obtained 2/70 (3%) clones showing a processed primer with RNPs prepared from HEK293T cells.It was previously reported that a nuclease activity in the RNP preparations could process primers before their extension Altogether these observations show that native L1 RNPs efficiently prime reverse transcription at DNA ending with 4–6 terminal matching nucleotides, although it can accommodate terminal mismatches with lower priming efficiencies.bona fide insertion sites. We designed 35 primers corresponding to previously published insertion sites recovered from new hL1 retrotransposition events obtained in cultured cells 20 oligonucleotide.L1 EN-mediated nicking at a consensus target site produces a 3′-OH DNA ending with four Ts in vivo without processing We observed that all sites are not equally extended see . The lev5′-TTTC-3′ and their respective levels of extension are LOU527<LOU538<LOU525, which roughly follows the number of Ts close to the 3′ end. This suggests a compensation mechanism allowing the extension of primers ending with suboptimal sequences.We categorized primers based on their potential of extension . To evaluate the number of terminal nucleotides that contribute to priming efficiency, we further correlated the priming efficiency with position-weighted T-density, taking into account a variable number of terminal nucleotides. The goodness-of-fit (R2) increases steadily up to 10 considered nucleotides and then reaches a plateau (2 = 0.8276).To address the significance of this phenomenon more quantitatively, we calculated for each oligonucleotide two parameters: (i) the density of Ts (number of Ts/length of the oligonucleotide), which simply reflects the abundance of Ts in the primer, and (ii) the position-weighted T-density, which is similar but the weight of each T is inversely proportional to the distance from the 3′ end  tail and the last 10 nucleotides of the target DNA plays a role in extension at the target site, the last 4 nucleotides being the most critical. Suboptimal primers with a mismatch in their last 4 nucleotides are extended with a lower efficiency, which can be partially compensated by increasing the number of Ts in the upstream sequence.To illustrate these findings, we propose that the four terminal bases of the primer, which overlap with the EN nuclease recognition sequence, act as a specific snap and the upstream six bases act as a weaker velcro strap . When th4. The velcro strap was considered as tightly fastened if the position-weighted T-density score of this region was at least half of its maximum value alue see . In agrein vivo, at the genomic level, L1 elements would more frequently insert at putative EN recognition sites with a closed snap and a tightly fastened velcro strap; and that a tightly fastened velcro would favor insertions as compared to similar sites with an open velcro. To test this model, we searched in the human reference genome (hg19) for the position of all potential EN targets: R/TTTT, which corresponds to a closed snap; or R/VTTT, R/TVTT, R/TTVT and R/TTTV, which correspond to open snaps . For each of them, we extracted the 10 nucleotides upstream of the nick position and categorized each on the basis of its snap/velcro status to obtain the exact frequency of each category in hg19. Then we extracted the exact insertion sites for all the L1HS polymorphic insertions present in dbRIP in vitro data, given a snap status, insertions are more frequent at sites with a tightly fastened velcro than with a loosely fastened velcro. Other studies have previously reported that T-richness extends beyond four nucleotides upstream of the cleavage site in vivo is consistent with the snap-velcro model at the genomic level, but it should also be stressed that, in vivo, other determinants are likely to influence L1 insertion profiles.A testable prediction of this model is that, An alternative pathway of L1 integration uses preformed double-stranded DNA lesions instead of EN-mediated cleavage. To determine whether the L1 RNP is able to directly initiate reverse transcription at blunt DNA ends, we designed model hairpins ending with four or six Ts at their 3′ terminus .18 primer , which depends on primer length see , is indi8 primer , lane 2,0 to H6). In contrast to reactions performed with blunt or 3′-recessed hairpin substrates, initiation of mL1 reverse transcription is easily detected as soon as the 3′ overhang reaches a length of 6 nt, as shown by the mL1-specific ladder which appears above 50 bp (Since L1 elements are believed to integrate into double-stranded genomic DNA and L1 RNPs can efficiently extend single-stranded oligonucleotides (see above), we reasoned that L1 RNPs might be able to prime DNA synthesis on double-stranded primers ending with a 3′ overhang. To test this hypothesis we designed model hairpins extended by a 3′ overhang of increasing size  partial sequence complementarity between the 10 terminal nucleotides of the target site and the L1 RNA poly(A) tail impact reverse transcription initiation  foitiation ; (iii) titiation ; (iv) thitiation . Based oitiation . This moin vitro provides detailed molecular insights into specific steps of the retrotransposition process. This is a useful complement to retrotransposition cellular assays, which offer a more global view of this mechanism. Nevertheless, a number of differences between the in vitro and in vivo situations, and between endogenously and ectopically expressed L1, should be emphasized. First, reverse transcription initiation is uncoupled from the cleavage of the target DNA, in primer extension assays such as LEAP or DLEA. Thus, we cannot completely exclude that L1 RNPs would utilize a different priming mechanism in the context of a L1 TPRT reaction. Likewise, it is possible that the detected activity results from a minor fraction of the RNPs, which can only extend exogenous primers. This situation is reminiscent of L1 reverse transcription initiation at existing DNA lesions as hypothesized for EN-independent integration events in vitro has been described In vivo, L1 elements carrying this endonuclease variant still integrate in extended T-rich sequences, which shows that additional factors other than the EN specificity contribute to L1 insertion profile in vivo. Our data suggest that primer-template complementarity might be one of these factors, by promoting the initiation of reverse transcription, but it is also very likely that additional partners or inhibitors influence L1 targeting in vivo, modulating or relaxing EN or RT specificity. Indeed, L1 insertions occasionally take place at sites that do not strictly follow the rules described here  versions of the human L1.3 element in a pCEP4 backbone (a kind gift of N. Gilbert) CNQYKNGNNALEKTRR-C) or human (N-CERNNRYQPLQNHAKM-C) ORF1p were synthesized and coupled to the KLH protein as a carrier. The first cysteine (underlined) is not present in the ORF1p sequence but was added for the coupling reaction with the carrier protein. KLH-coupled peptides were used to immunize rabbits (Eurogentec). For immunoblotting the mORF1p antiserum (SE-0560), the hORF1p antiserum (SE-6798), and the S6 protein antibody  were used at a dilution of 1∶2000.Peptides corresponding to the C-termini of mouse (N-2. Eluted oligonucleotides were precipitated with ice-cold ethanol (3v). After centrifugation for 30 min at 4°C at 16'000 g, the pellets were washed with 70% ethanol, air-dried and dissolved in 10 mM Tris-HCl pH 8.0, 1 mM EDTA.One hundred micrograms of each lyophilized oligonucleotide was dissolved in 10 µl of 98% deionized formamide, 1 mM EDTA, 0.01% (w/v) xylene cyanol and 0.01% (w/v) bromophenol blue and resolved in 10% polyacrylamide-urea denaturing gels. Full length oligonucleotides were visualized by UV shadowing, excised from the gel and eluted overnight at 37°C in 0.3 M sodium acetate, 0.1% SDS and 10 mM MgCl6 cells per 10 cm Petri dish. Twenty-four hours after plating, the cells were transfected with 24 µg of plasmid DNA (see plasmids above) per dish using the calcium phosphate method. Growth medium was changed 5 hours later. One day post-transfection, cells were split into two plates in growth medium supplemented with 1.5 µg/mL puromycin  or 100 µg/mL hygromycin . Cells were collected 4 days post-transfection by trypsinization, pooled and washed in PBS. Cell pellets were lysed in 500 µL of CHAPS lysis buffer  CHAPS, 10% (v/v) Glycerol, supplemented before use with Complete EDTA-free protease inhibitors cocktail (Roche) and 1 mM DTT). After incubation at 4°C for 15 min, cell debris was removed by spinning down extracts at 4°C for 10 min at 16'000 g. Supernatants were transferred to clean tubes and 500 µL of lysis buffer were added to each of them.L1 RNPs were produced in HEK293T cells grown in Dulbecco's Modified Eagle Medium  containing 2 mM L-Glutamine, 4500 mg/L D-Glucose, 1 mM Sodium Pyruvate, 10% (v/v) fetal bovine serum (Life Technologies) and 100 units/mL penicillin/streptomycin (Life Technologies). Cells were plated at 3×102, 1 mM DTT and Complete EDTA-free protease inhibitors cocktail (Roche). For each sucrose cushion, 1 mL of cell lysates, prepared as described above, was used. Samples were centrifuged for 2 h at 178'000 g at 4°C and the pelleted material was resuspended in 100 µL H2O. Total protein concentration was determined by Bradford assay (Biorad). The samples were diluted in 50% (v/v) glycerol, quick frozen in liquid nitrogen and stored at −80°C until use.L1 RNPs were prepared as previously described Protein A-Sepharose beads (Sigma) were blocked overnight at 4°C in PBS containing 0.5 mg/mL of bovine serum albumin (BSA) and washed twice in 1 mL of IP buffer . Eight microliters of preimmune or anti-mORF1p serum were bound to 70 µl of blocked beads for 3 h at 4°C. For each immunoprecipitation, 200 µL of L1 RNPs (2 µg/µL) were diluted 1∶1 (v/v) in IP buffer. The RNPs were precleared with blocked beads for 1 h at 4°C and incubated for 3 h at 4°C with antibody-bound beads on a rotating wheel. After 4 washes in IP buffer, the bead slurry was split equally into 7 tubes (6 for RT reactions and 1 for immunoblotting). Beads were pelleted for 5 min at 4°C at 750 g, supernatants were removed and the RT reaction mixture was directly added to the beads (see below).2, 10 mM DTT, 0.05% (v/v) Tween-20 and 10 µCi of α-32P-dTTP . In reactions using the Avian Myeloblastosis Virus RT , the RNPs were replaced by 0.04 U of AMV RT and 250 ng of poly(rA) template (Roche). Reactions were stopped by the addition of 8.3 mM EDTA and 0.83% SDS final. Trace amounts of a 32P-labelled 14- or 30-mer DNA oligonucleotide were added as recovery control (noted RC (14) or RC (30) in the figures). Products were purified by phenol-chloroform extraction and ethanol precipitation with 10 µg of glycogen as a carrier and 0.1 mM sodium acetate [pH 5.2]. DNA pellets were resuspended in 98% deionized formamide containing 10 mM EDTA, 0.02% (w/v) xylene cyanol and 0.02% (w/v) bromophenol blue, heated to 95°C for 5 min, and analyzed on 13% polyacrylamide-urea sequencing gels. After drying, gels were exposed to a PhosphorImager screen.Reverse transcriptase assays were carried out for 4 min at 37°C in 25 µL reactions containing 2 µg of RNPs, 400 nM of primer, 50 mM Tris-HCl [pH 7.5], 50 mM KCl, 5 mM MgClFor primers used in For gel or spot quantification, the reaction without primer obtained with a given RNP preparation was used as background and was subtracted from the reaction with primers. Only the signal above the primer size was quantified for the hairpin oligonucleotides.32P incorporation was RNase sensitive  as triphosphate derivatives were obtained from Biocentric. They were added to reactions at a final concentration of 10 µM  Tween-20, 20 U RNasin (Promega), 200 µM dNTP, and 0.4 µM LEAP primer. Eventually, unextended primers were eliminated through an S-400HR size-exclusion spin column . Reverse transcription products (1 µL of the LEAP reaction) were PCR-amplified in 50 µL reactions containing 1 U of Platinum Taq DNA Polymerase (Life technologies), 0.2 µM of primers LOU851 and LOU312, 200 µM dNTP, 3 mM MgCl2 in the Platinum Taq buffer. A first step at 94°C for 2 min was followed by 35 cycles of . The final extension was at 72°C for 5 min. PCR products were analyzed by 2% agarose gel electrophoresis in 1x TBE. Gels were stained by SYBR Safe (Life technologies) or ethidium bromide. LEAP products were gel-purified with a gel extraction kit (Macherey Nagel) and cloned into the pGEM-T-easy vector (Promega), according to manufacturer's protocol. Clones from isolated colonies were sequenced by GATC. Regions with low quality (Phred<Q20) were trimmed or filtered out using Geneious 5.LEAP was performed as previously described 2 and 5 mM DTT. Primer pairs used for PCR were LOU851/LOU312 (mOrfeus or L1.3) or LOU852/LOU312 (GAPDH). PCR products were resolved by 2% agarose gel electrophoresis in 1x TBE.Total RNA was extracted from 30 µg of L1 RNP using TRIzol extraction (Molecular Research Center Inc) following the manufacturer's instruction. RNA was resuspended in 20 µL of milliQ water and quantified by Nanodrop. One microgram of RNA was digested by 1 U of RNase-free RQ1 DNase (Promega) in 10 µL reaction in the manufacturer's buffer at 37°C for 30 min. DNase was heat-inactivated for 10 min at 65°C. Then, cDNA synthesis was performed at 50°C for 1 h in 20 µL reactions containing 6 µL of the DNase reaction, 200 U of SuperScript III Reverse Transcriptase (Life technologies), 500 µM dNTP, 50 pmol of RACE primer, 40 U RNAseOUT (Life technologies), 50 mM Tris-HCl [pH 8.0], 75 mM KCl, 3 mM MgClT-density is calculated by dividing the number of Ts in the oligonucleotide by the length of the oligonucleotide. The position-weighted T-density gives more weight to Ts which are close the 3′ extremity of the primer. The weight is inversely proportional to the distance from the 3′ end.The For example:position-weighted T-count equal to:Primer LOU519 has a position-weighted T-count equal to:Primer LOU541 has a position-weighted T-density of a given primer is calculated by dividing the position-weighted T-count of this primer to the maximum position-weighted T-count. Thus the position-weighted T-density of LOU519 is equal to 2.23/3.60 = 0.62 and the position-weighted T-density of LOU541 is equal to 3.60/3.60 = 1The position-weighted T-count for the upstream 6 nucleotides (velcro region) and we divided it by the maximum value (1/5)+(1/6)+…+(1/10) = 0.84563492 to obtain the velcro position-weighted T-density. We consider a velcro as fastened if its position-weighted T-density is ≥0.5  and opened otherwise.The snap is considered open if the 4 terminal nucleotides contain a non-T nucleotides and closed if the last four nucleotides are 4 Ts. We calculated a All putative integration sites with a perfect or degenerate EN recognition sequence  were recovered from both strands of the reference human genome (hg19) or from its repeatmasked version (hg19 RM). For each putative EN site, snap and velcro status were defined as described above. The C++ program used to achieve this task is available in Protocol S1. Polymorphic L1 insertions were extracted from dbRIP Figure S118 primer extension by hL1 RNP. (D) Formation of long cDNA species upon addition of all four dNTPs. Reactions were performed with hL1 RNPs in presence of α-32P-dTTP and a (dT)18 primer, with (lanes 3 & 6) or without (lanes 1–2 & 4–5) cold dATP, dCTP and dGTP .Additional characterization of the L1 RNP RT activity by DLEA. (A) RNA-dependent DNA polymerase activity of L1 RNPs. Murine L1 RNPs were incubated for 1 h at 37°C in the presence (lane 3) or in the absence (lane 4) of RNases before the start of the reaction. (B) RT inhibitors prevent primer extension by L1 RNPs. Reactions were performed with mL1 RNPs in the presence of thymidine analogs , or in the presence of water as a negative control (lane 2). (C) Time-course of (dT)(TIF)Click here for additional data file.Figure S232P-dTTP. RC denotes a 14 nt recovery control added after the reaction but before DNA purification. The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Quantification of primer extension (% Activity) was relative to levels of extension obtained with oligo(dT)18. Primers are identical to The murine L1 RNP preferentially extends primers ending with at least 4 Ts. DLEA showing the extension of single-stranded primers by mL1 RNPs in the presence of α-(TIF)Click here for additional data file.Figure S3LEAP with hL1 RNPs and mismatched primers. (A) Primers with terminal mismatches. LEAP was performed with RNPs prepared from hL1-transfected cells (top panel), from vector-transfected cells (middle panel), or without RNPs (bottom panel). Primers are identical to those used in (TIF)Click here for additional data file.Figure S432P-dTTP. Note that the products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities  Absence of extension by hL1 RNPs of double-stranded primers with blunt or 3′-recessed end in the presence of α-(TIF)Click here for additional data file.Protocol S1Source code of the software used to find putative endonuclease sites in the human genome and to calculate their associated snap/velcro scores.(GZ)Click here for additional data file.Table S1List of oligonucleotides used in this study.(XLSX)Click here for additional data file.Table S2Data used to calculate genomic enrichment of L1 insertions depending on the snap-velcro status of the target. The table sheets are the following: (hg19) For each potential L1 EN target site present in hg19, the snap status was defined and the position-weighted A density was calculated. Sites with position-weighted A density equal to or above 0.5 were considered as having a closed velcro strap. (hg19 RM) Same as above but with a repeatmasked hg19 reference genome. (dbRIP sequences) L1HS dbRIP entries used in (XLSX)Click here for additional data file.


Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with “higher” metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges:  Amphimedon queenslandica revealed that intron position and phase are conserved relative to other metazoans Trichoplax adhaerens are not conserved in human A. queenslandica, which may indicate that metazoan complexity correlates with an increasing number of miRNAs Nme6 gene, but not in the sponge ortholog, contain miRNAs also supports that thesis cis-regulatory architecture in promoters of RPGs appears not to be drastically changed from sponge to human A. queenslandica RPG introns are similar in many structural characteristic to “higher” metazoans. Furthermore, the sponges A. queenslandica and S. domunucula show as much conservancy of RPG intron positions between each other as between themselves and human. However, when we compared RPG introns with three species from the genus Suberites of sponges, there was a discrepancy in snoRNA pools between the genus Suberites and A. queenslandica. Even though the intron positions in the Suberites genus are conserved, there are significant differences in certain RPG introns which indicate that they are dynamic even on these shorter evolutionary time scales.The ribosome is a protein-RNA complex, fundamentally the same in all three domains of life, and is the crucial cell component for protein synthesis. X-ray crystal structures of ribosomal subunits were used to determine ribosome structure and function in detail A. queenslandica expressed sequence tags (ESTs) encoding homologs of human RPs TBLASTN  was used. The set of sponge RPGs was obtained using the NCBI WGS (whole genome shotgun) Trace Archive as well as the assembled draft genome available at http://spongezome.metazome.net/cgi-bin/gbrowse/amphimedon/http://codonw.sourceforge.net/), while the GC content of the whole genome was calculated using geecee from the EMBOSS suite Homo sapiens, Strongylocentrotus purpuratus, Drosophila melanogaster, Caenorhabditis elegans, Nematostella vectensis, Trichoplax adhaerens, Monosiga brevicollis, Saccharomyces cerevisiae and Arabidopsis thaliana). One RPG from T. adhaerens, four from N. vectensis and ten from M. brevicolis were assembled manually from data available on the NCBI Trace Archive to give a total set of 55 orthologs from nine organisms. Due to the lack of a fully annotated set of RPGs from the nine organisms mentioned, it was not possible to analyze the entire set. RPGs analyzed are listed in http://www.psc.edu/biomed/genedoc). MEME was used for searching for over-represented motifs in RPG introns http://snoopy.med.miyazaki-u.ac.jp/) and the snoRNA-LBME database Suberites domuncula RPGs, the corresponding and neighboring introns were sequenced. All primer sequences used in this study are given in Suberites ficus and Suberites pagurorum from genomic DNA isolated as previously described S. domuncula were cut into pieces, frozen in liquid nitrogen and ground to a fine powder. Approximately 3 µg of small RNAs were obtained from 260 mg of tissue powder by the mirPremier microRNA Isolation Kit (Sigma), according to the manufacturer's protocol for plant tissue. Polyadenilation of 1 µg of small RNAs was achieved by incubation with 5 U of the E. coli Poly(A) Polymerase (BioLabs) for 15 minutes at 37°C. All 12 µl of the poly(A) tailing reaction mixture was then reverse transcribed using the SuperScript II Reverse Transcriptase (Invitrogen) and a modified poly-d(T) primer (modpolydT). The resulting cDNA was diluted to 100 µl and each PCR was performed on 3 µl of cDNA using the HotStarTaq DNA Polymerase (Qiagen), a universal reverse primer (uni) and forward primers specific for predicted snoRNAs. The products were cloned into the pGEM-T vector (Promega). Positive clones were sequenced using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit and T7/pUC primers.To identify A. queenslandica (AQ) genome, and the gene structure for 78 of them was completely ascertained. The RPS14 gene was not completely determined due to the absence of WGS sequences indispensable for assembling one long intron. This gene was not considered in calculating the average values shown in N. vectensis and 73 from placozoan T. adhaerens were used as control. Sponge AQ RPGs contained an average of 4.01 introns. The RPP0 gene had the largest number of introns - 10, while the only gene without introns was RPL35 (RPS27 gene (2263 bp), and the shortest one was the first intron of the RPS21 gene (37 bp). Human RPGs have significantly longer introns of 760 bp on average RPS9 gene. Most of the introns found between translational start and stop codons, were phase 0 (52%), 27% were phase 1 and 21% were phase 2. These results support the so-called “50/30/20 rule” of intron phase distributions. It has been found that across many studied organisms, approximately 50% of introns are phase 0, 30% are phase 1 and 20% are phase 2 S. domunucula (SD) RPGs have a preference for C- and G- ending codons Full-length cDNA sequences coding for 79 RPs were identified in the marine sponge as RPL35 . In 78 cas RPL35 , which iH. sapiens (HS), S. purpuratus (SP), D. melanogaster (DM), C. elegans (CE), N. vectensis (NV), T. adhaerens (TA), M. brevicollis (MB), S. cerevisiae (SC) and A. thaliana (AT). The total number of analyzed introns within the coding regions of the RPGs in ten organisms was 1491  and S. pagurorum (SP) which produced a candidate set of 9 C/D box snoRNAs and 2 H/ACA snoRNAs in each of the sponge  and invertebrates  and checked for the presence and the position of the SNORD100 ortholog. There is an obvious tendency of this snoRNA to colonize the RPS12 gene from basal metazoans to “higher” vertebrates , found in the second intron of the RPL7A gene. It is predicted to guide the 2′-O-ribose methylation of 28S rRNA cytosines at position 2338 and 2352 in human RPP0 gene and is most similar to human SNORD83A/SNORD83B (U83A/U83B) found in the fifth and seventh intron of the human RPL3 gene, respectively  of this snoRNA are still unknown RPL13A gene. While AQ possesses only one copy, in SD, SF and SP this snoRNA was found duplicated in the neighboring intron. The SD copies are 94% identical to each other, while one shares only 48% and the other 49% identical nucleotides with those from AQ. Although overall not well conserved, all essential snoRNA elements and target sites are maintained  and S. pagurorum (SP). The total of 126 RPG introns examined  of these rRNA's target sequences are well conserved in sponge, three targets of three snoRNAs are unknown, two targets are not determined due to incomplete sponge 28S rRNA sequences, and seven are poorly conserved. Presence of the majority of target rRNA sequences lead us to seek out whether these snoRNA orthologs are nested somewhere else in the genome of AQ. We checked for orthologs of 19 C/D box snoRNAs with conserved target rRNA. Six conserved orthologs were found see . This inFrom all these results we can conclude that sponge RPG introns share many structural characteristics with “higher” metazoans. These similarities are probably important because RPG introns function as carriers of snoRNAs. Intensive mobility of snoRNAs is probably the reason why sponges from different genera show discrepancy in snoRNA pools in orthologous RPG introns. Their mobile nature is also a plausible reason for almost equal RPG intron positions' conservation between sponges from different genus as between sponges and human, which indicates their more important role in diversification and enrichment of transcriptomes through mechanisms such as intron/exon gain/loss. Within the same genus large insertions in orthologous RPG introns were detected. Mechanisms of intron dynamics on this shorter evolutionary time scale is influenced by factors not correlated with mobility of snoRNAs.Figure S1Percentage change from the consensus sequence of RPG introns from three species of the Suberites genus. Introns (4L28 through 2L14 on the y axis) from S. domuncula (SD), S. ficus (SF) and S. pagurorum (SP), were aligned and the percent change from the resulting consensus sequence is shown per species. The average column shows the average percent change per intron for all three species. The last set of bars is the percent change from the consensus for the concatenated sequences of all introns from a species (CONCAT on the y axis).(TIF)Click here for additional data file.Figure S2Human (HS) RPG-sited snoRNAs identified in sponge (AQ) introns of non-RP genes. All essential snoRNA elements and methylation (Me) guide sites are designated.(TIF)Click here for additional data file.Table S1Characteristics of 55 RPGs from seven model organisms.(DOC)Click here for additional data file.Table S2Sequences of primers used in this study.(DOC)Click here for additional data file.Table S3Characteristics of RPG introns in three basal metazoans.(DOC)Click here for additional data file.Table S4Comparison of 55 RPGs in ten organisms.(DOC)Click here for additional data file.Table S5Percentages of amino acid identity  and overall similarity  extracted from GeneDoc.(DOC)Click here for additional data file.Table S6Percentages of intron positions shared among organisms.(DOC)Click here for additional data file.Table S7The sequences of the C/D and H/ACA box snoRNAs identified in RPG introns of sponges A. queenslandica (Aq), S. domuncula (Sd), S. ficus (Sf) and S. pagurorum (Sp). Conserved snoRNA elements are boxed and predicted methylation guide sites are shaded. Experimentally verified snoRNAs are marked with asterisk (*). Lowercase letters indicate nucleotides that were not sequenced due to the snoRNA cloning strategy. Letters in brackets indicate nucleotides which do not belong to snoRNA, although they were predicted by snoSeeker.(DOC)Click here for additional data file.Text S1126 introns sequences from three Suberites species: S. domuncula (SD), S. ficus (SF) and S. pagurorum (SP).(TXT)Click here for additional data file.


Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways. Post-transcriptional regulatory function arises through the retention of specific intronic, nonprotein-coding sequences within the cytoplasmic mRNA. We call such sequences Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs). For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons and proper channel protein localization to dendrites [Recently, several studies have revealed a new dimension to the diversity of transcripts , for insendrites . Intron endrites . A retaiendrites . In a reendrites . These sHere we performed a transcriptome-wide survey for intronic sequence retention using subcellular RNA sequencing of dissected neurites from rat and mouse hippocampal neurons. Neurons are highly polarized cells with spatially distinct dendrites and axons that allow mechanical isolation of cytoplasmic RNA through microdissection. Strictly speaking, our subcellular dissections do not distinguish dendrites and axons but in these neurons the axonal compartment is less than 5% of the volume of dendritic compartments; therefore, we will call our samples dendritic samples from hereon. In addition to cytoplasmic RNA from dendrites, for contrast we also carried out single cell RNA sequencing of individual cell somas, which also contains some heterogeneous nuclear RNA (hnRNA). These datasets were augmented by single cell sequencing from mouse Brown adipose tissue (BAT)  and mousWe performed a comprehensive transcriptome wide survey of the prevalence of CIRTs in rat and mouse dendritic population. Two batches of dendritic mRNA from primary rat hippocampal neurons and three batches of dendritic mRNA from primary mouse hippocampal neurons, each consisting of 150–300 individually dissected dendrites were independently aRNA amplified [Of the reads mapping to intronic regions, only the uniquely mappable reads by paired-end Bowtie alignment  were couGuttman et al [ab inito reconstruction of these transcriptomes [First, each intron with putative cytoplasmically retained sequence was also filtered for overlap with predicted genes using the tools GENSCAN  and TWINan et al  reportedan et al  for overan et al . Since triptomes . We founAfter processing through these steps to minimize other origins of intronic sequences in the mRNA sequences, we found ~44% rat genes and ~59% of the mouse genes in dendritic samples showing intron sequence read evidence consistent with the hypothesis of CIRTs . For the5’ to 3’ introns of a gene model . We chose to have this larger distance threshold in order to account for the reads arising from repetitive sequences that tend to be enriched in intronic regions and may not be uniquely mappable and also to be conservative in the number of independent putative CIRTs. This distance is also roughly equal to two back-to-back paired-end reads. Of all the possible introns within the genes expressed, we found that cytoplasmic retention occurs in only a small fraction of the introns (13-16%)  in rat ane model . We alsone model . It is lne model . Also, ~The retained intronic contigs in rat dendritic samples had a mean length of 363 nt while in mouse dendritic samples the mean length was ~353 nt . The mear = 0.001, P-value = 0.9, Sample size = 3947). Another factor that may affect the resulting length of the contigs is the multiple rounds of aRNA amplification steps in our protocol, which can cause a shortening of the transcripts due to random primer binding for second-strand synthesis in each subsequent cycle. However, in rat dendritic samples, about 3-5% of the contigs had much longer lengths in the range of 1000 bp to 5000 bp and even for these long contigs there was no correlation between the contig length and maximum pA tract contig length  and they arise in almost equal proportions from the 5’ and 3’ introns of the transcript. In sum, the specific characteristic length distribution and selectivity of introns with cytoplasmically retained sequence seem to display distinct signatures of specific processing for CIRT biogenesis independent of the aRNA procedure. A visual representation for few randomly selected CIRTs has been created in UCSC Genome Browser and is provided in the supplementary -T7 primers which recognizes pA-tracts including those from intronic regions. To assess whether the contig length distribution is related to chance occurrence of pA tracts within introns, we first constructed contigs of pA tracts by clustering pA sequences at least 9 bp long and separated by less than 500 bp. Of all the introns with cytoplasmically retained sequence in rat dendritic samples, ~56% had at least one internal pA tract. For these introns, there appeared to be no correlation between the average pA tract contig length and the average intronic contig length  from the transcripts present in the tissue sample were found to give rise to intronic contigs. However, the contig lengths were significantly longer (mean length ~ 1500 bp). . We founAfter having analyzed the properties of putative CIRTs from the carefully dissected dendrites that are free of nuclear hnRNA, we turned our attention to the whole single cell. Single cell transcriptomes are also expected to contain mostly cytoplasmic RNA see  but sincde novo. By considering only the high quality junctions reported by RUM which have known splice signals and are crossed by at least one uniquely mapping read with at least 8 bp on each side of the junctions, we found several novel intron-intron junctions with canonical or known non-canonical splice signals in the both rat and mouse dendritic as well as soma samples . Additionally we also analyzed the genomic DNA from rat hippocampus to verify whether the putative spliced intronic region is in fact present in the genome. The rat strain used for obtaining the mRNA and genomic DNA was Sprague-Dawley which is the same strain from which we got the dendritic samples. Even then there is a potential for copy number variations between individual animals that cannot be ruled out. Of all the novel intron-intron junctions in rat samples, we selected the junctions for experimental validation based on the following criteria: i) junctions for which there was evidence for sufficiently long cytoplasmic retained intronic sequence surrounding the junction with high complexity sequences for primer design, ii) above median expression of the transcript in rat hippocampus tissue, iii) length of the intron containing the junction is shorter than 20,000 bp, and iv) junctions with spliced out length of >= 100 bp. This left us with 78 putative rat intron-intron junctions of which we selected 7 junctions that were detected in either rat soma or dendrites . For twoLastly, we also surveyed the EST database available on UCSC Genome Browser  to assesHaving observed a widespread presence of CIRTs in our neuronal samples, we were curious about their presence in other cells. To do so we analyzed some non-neuronal single cell datasets, namely five independently collected single cells from mouse brown adipose tissue (BAT)  and eighFor the whole single cell samples: mouse soma, BAT and cardiomyocyte samples, we found the average pair-wise overlap among the genes giving rise to CIRTs to be ~44%. On average, when two cells have retained intronic sequences in the same gene, in ~23% of the cases the retained sequence was from identical introns. A gene may show shared evidence of retained introns in various different cell types, but the particular retained introns may differ. For example, one such gene was the MLH3 gene which encodes the DNA mismatch repair protein Mlh3. This gene is associated with mammalian microsatellite instability . This geGiven the widespread prevalence of CIRTs as detected by our analysis we investigated the functionality of the genes giving rise to CIRTs. It is possible that for majority of genes with CIRTs, the CIRTs comprise a minor transcript form while majority of the transcripts do not contain CIRTs as shown in . These m There were 707 genes in rat dendritic samples with intronic read count >= 256 from at least one retained intron. We performed GO analysis on this set using DAVID Bioinformatics Resources 6.7 ,31. AmonAs reported in the above, as part of our filtering step we analyzed for overlap of CIRTs with ncRNAs. We found <2% of the retained introns in mouse and rat dendrite samples to have the Rfam  annotateIn this paper, we use RNA sequencing data from micro-dissected cytoplasmic compartments to show evidence supporting genome-wide prevalence of cytoplasmic intron-sequence retaining transcripts (CIRTs). Surprisingly, up to 59% of expressed genes in rodent neurons showed evidence of sequence reads from intronic sequences. Similar proportions were also seen in mouse Brown Adipose Tissue (BAT) cells and mouse cardiomyocytes. Support for this genome-wide presence of CIRTs come from several sources. First, our RNA populations for the neuronal cells were derived from carefully dissected non-nuclear dendritic segments with little potential for contamination from hnRNA. This fact was supported also by distinct distribution of intronic RNA sequences in contrast to the distributions seen from single cell soma sequencing and whole tissue sequencing. Second, we used multiple computational filters to remove potential effects of cryptic exons, ncRNA, etc. Third, previous work from our lab showed extensive evidence for CIRTs using PCR cloning, in situ hybridization, and RNA sequencing leading to the hypothesis that CIRTs were prevalent across the transcriptome . In addiIf CIRTs play a role in post-transcriptional cell physiological processes, we hypothesize that non-random subsequences of introns are specifically retained rather than retaining any introns of a given gene. Many mammalian introns are extremely large and the median size of mouse and rat introns is ~1300 bp . The contigs assembled from our RNA sequencing suggest typical CIRT size in the range of 350-400 bp, suggesting that the CIRT region of a transcript is processed in the nucleus to a specific subsequence. This size range data is supported by existence of a sharp second mode around 300 bp in intronic sequences from normal tissue RNA sample. In addition, when we find evidence of cytoplasmic intronic sequences for a given gene, typically the sequence evidence is present for only a specific intron rather than all of the gene’s introns. Finally, we found numerous examples of intron-intron junctions surrounding the CIRTs and carried out experiment validation for seven putative intron-intron junctions. We were able to show evidence of spliced product for five of these junctions. We note that our annotation of the intron-intron junctions were limited to known canonical and non-canonical splice signals and if we were to expand the definition to novel junction types, we may have considerably larger annotation of intron-intron processing. Thus from these data we hypothesize that at least some proportion of transcripts for many genes in the genome are alternatively processed during splicing to retain specific subsequences of intronic sequences. Recently, there have been other findings that also lead to the speculation of alternate splicing mechanisms giving rise to a diversity of transcripts not thought to exist previously. For instance, the detection of chimeric RNAs comprising of sequences from different genic boundaries, even those that are on different chromosomes, suggest the involvement of an alternate splicing machinery [The functional roles of CIRTs are uncertain at this stage. In Buckley et al. , we demoIn conclusion, we hypothesize that retention of subsequences of introns involves specific alternative splicing pathways and such sequence retention is wide spread for genes in the mammalian genome. We speculate that these CIRTs play a role in post-transcriptional regulation and possible RNA-based signaling after cytoplasmic processing. Overall, the presence of CIRTs increases the complexity of the transcriptome and CIRT modulation may present another pathway for epigenesis of cellular complexity and play a role in neuronal plasticity.Hippocampi were harvested from embryonic day 18 rat (Sprague-Dawley) and mouse (C57BL/6) pups and dispersed and plated at 100,000 cells per ml of neurobasal medium and B27 (Invitrogen). Neurons are grown in culture for 14 days on 12 mm round German Spiegelglas coverslips (Bellco) coated with poly-L lysine (Peptide Institute).Independent samples of two sets of approximately 150-300 dendrites and 3 cell soma from primary rat hippocampal neurons and three sets of approximately 150-300 dendrites and 3 cell soma from mouse hippocampal neurons were harvested by mechanical isolation and used as template material for aRNA amplification (as described in ).de novo junctions. The number of reads in each library and alignment statistics are provided in Paired-end libraries were constructed from fragmented dendritic and soma aRNA material and sequenced by using Illumina II short-read technology. Paired-end libraries were also constructed for total RNA from whole hippocampus tissue and sequenced using Illumina II short-read technology. The read length is 50 bp. Reads were trimmed for adapter sequence and poly-A, poly-T and then aligned to the Rat (rn4) and Mouse (mm9) genome by using the RUM pipeline . The RUMThe BAT cells were collected in the lab of J. Eberwine . BrieflyThe cardiomyocytes were collected in the lab of B. Kuhn. Briefly, embryonic cardiomyocytes were isolated from the ventricles of 14.5 day old of a hemizygous Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) embryo . The carRNA from these single cells were collected, amplified and sequenced as described above.The collection of the primary cultured cells utilized animal by-products protocol, "Genome Biology of Single Neuron Funciton and its Modulation with Age" under University of Pennsylvania IACUC protocol #803321 . Animals were sacrified under University of Pennsylavania IACUC protocol #804867, "Molecular Biology of Single Aging Neurons and Glia" , but the sacrifice of the animals were independent of the work reported in this paper. The cardiomyocytes were collected under Harvard University IACUC protocol 12-05-2169R, "Mechanisms of myocardial regeneration" . All protocols were approved by University of Pennsylvania and Harvard Office of Regulatory Affairs and IACUC committee.Primers were designed from the intronic region surrounding the putative intron-intron junction for which we had sequence evidence for cytoplasmic retention . cDNA waFor every intron in the dendritic samples that showed evidence for cytoplasmic intron sequence retention, we selected another intron randomly such that it had a similar length and GC content (within a range of 5%) as well as an identical internal pA-tract length and belonged to a gene that had no cytoplasmic intron sequence retention. In this way we were able to construct a background intron set size matched with the set of retained introns for both mouse as well as rat. File S1Figure S1, Fraction of CIRTs as a function of reads mapping to the retained intronic contigs. Figure S2, Fraction of retained introns/gene as a function of total number of intronic reads/gene. Figure S3, Fraction of intronic contigs falling within each one-third region of the introns. Figure S4. Visual representation of the reads mapping to randomly picked genes that give rise to putative CIRTs. Figure S5, Experimental validation by RT-PCR of seven putative intron-intron junctions from rat dendrites and soma. Table S1, Position of Rat and Mouse dendrites retained introns within the gene (for genes with at least 5 introns). Table S2, Retained introns in Rat and Mouse dendrites with internal pA-tract of varying lengths.(DOC)Click here for additional data file.Table S3List of novel intron-intron splice junctions.(XLS)Click here for additional data file.Table S4Experimentally validated intron-intron splice junctions.(XLS)Click here for additional data file.Table S5Gene ontology analysis results.(XLSX)Click here for additional data file.Table S6Next generation sequencing alignment statistics.(XLS)Click here for additional data file.Table S7List of primers used for experimental validations.(XLS)Click here for additional data file.


Although intron loss in evolution has been described, the mechanism involved is still unclear. Three models have been proposed, the reverse transcriptase (RT) model, genomic deletion model and double-strand-break repair model. The RT model, also termed mRNA-mediated intron loss, suggests that cDNA molecules reverse transcribed from spliced mRNA recombine with genomic DNA causing intron loss. Many studies have attempted to test this model based on its predictions, such as simultaneous loss of adjacent introns, 3'-side bias of intron loss, and germline expression of intron-lost genes. Evidence either supporting or opposing the model has been reported. The mechanism of intron loss proposed in the RT model shares the process of reverse transcription with the formation of processed pseudogenes. If the RT model is correct, genes that have produced more processed pseudogenes are more likely to undergo intron loss.In the present study, we observed that the frequency of intron loss is correlated with processed pseudogene abundance by analyzing a new dataset of intron loss obtained in mice and rats. Furthermore, we found that mRNA molecules of intron-lost genes are mostly translated on free cytoplasmic ribosomes, a feature shared by mRNA molecules of the parental genes of processed pseudogenes and long interspersed elements. This feature is likely convenient for intron-lost gene mRNA molecules to be reverse transcribed. Analyses of adjacent intron loss, 3'-side bias of intron loss, and germline expression of intron-lost genes also support the RT model.Compared with previous evidence, the correlation between the abundance of processed pseudogenes and intron loss frequency more directly supports the RT model of intron loss. Exploring such a correlation is a new strategy to test the RT model in organisms with abundant processed pseudogenes. The loss of spliceosomal introns in eukaryotic evolution has been well documented -11. HoweSaccharomyces cerevisiae, recombination between a reverse transcript and its chromosomal homologue was detected [Drosophila and mammals showed that genes expressed in the germline have a higher frequency of intron loss [Arabidopsis [Although the RT model of intron loss is widely cited, it is still controversial and far from being universally accepted. In the yeast detected ,19. Howedetected -26. By cdetected -29. Secodetected ,25,30-33detected ,27,34-36detected ,38. In tdetected ,24. ThirIn this paper, we describe a new approach to test the RT model. In the RT model, the frequency of intron loss depends on the abundance of reverse transcripts, which are thought to be determined by RT activity and the affinity of mRNA templates for RT. Processed pseudogenes (PPs) are the byproducts of RT encoded by retrotransposons . The actAmong the 16,241 intron-containing orthologs between mice and rats, we identified 148,176 conserved introns and 937 unique intron positions that might refer to possible intron losses or gains. By consulting the orthologous genes in seven outgroup mammal species .In rats, we found that 27.7% IL genes were parental genes of PPs. By contrast, among 8,410 NIL genes, only 7.4% were parental genes of PPs. Fisher's exact test showed that the proportion of parental genes of PPs in IL genes was significantly higher than that of NIL genes . These results showed that intron loss frequency in rats is positively correlated with the abundance of PPs.Also in rats, we found that the parental genes of PPs had a higher rate of intron loss than other genes. On average, each parental gene of PPs lost 0.041 introns whereas each non-parental gene lost only 0.008 introns (Mann-Whitney The PPs produced by genes with short mRNAs are more likely to be detected  and geneP < 10-7). Genes with larger numbers of PPs had fewer extant introns.If genes with more PPs have more introns, they would be more likely to lose introns just by statistical chance. However, we observed a significant negative correlation between the number of extant introns and the abundance of PPs . Similar results were obtained by comparing the IL genes and NIL genes of mice . Further analyses using a larger sample size or high coverage data of mRNA decay rate are required to reach a definite conclusion.Pavlicek et al.  also fouet al.  and the -4 indicated that adjacent introns tend to be lost simultaneously  . In totaet al. [P = 0.32; rats, 12 out of 86 versus 28,914 out of 148,176, P = 0.24). Therefore, we found evidence of genomic deletion but no evidence of the NHEJ model of intron loss in mice and rats.Besides the evidence supporting the RT model of intron loss, we also searched for evidence of genomic deletion and the NHEJ model. First, in our dataset of intron loss, there were seven imprecise intron losses in mice and rats  and the formation of PPs. IL genes and parental genes of PPs share properties that may facilitate reverse transcription, such as being translated on free cytoplasmic ribosomes. More importantly, we found a positive correlation between the frequency of intron loss and the abundance of PPs. Our results strongly indicate that reverse transcription is a necessary step in intron loss. IL genes in mammals were found to be highly expressed . We alsoBeside direct recombination of cDNA with genomic DNA, the RT model has another sub-model: recombination or gene conversion of genomic DNA by intronless PPs . If a PPThere is another possible but less likely explanation for the correlation between intron loss frequency and PP abundance. Highly expressed genes may be more likely to lose introns to reduce metabolic load and the probability of mis-splicing than lowly transcribed genes. Because highly expressed genes have generated more PPs, the intron loss frequency and PP abundance are linked together superficially by high expression level. The metabolic load of introns was previously supposed to be a selective force to intron length reduction in highly expressed genes -60. HoweDrosophila and Arabidopsis thaliana using the datasets of intron loss previously published [Drosophila or Arabidopsis (0.23% for Drosophila and 0.73% for Arabidopsis). Fisher's exact tests showed that the differences between IL genes and NIL genes are not significant in either Drosophila or Arabidopsis (P > 0.6 for both cases). Considered just from this result, it seems that the RT model is not the major mechanism of intron loss in either Drosophila or Arabidopsis. However, previous studies suggested the RT model might be the major mechanism of intron loss in Drosophila but not in Arabidopsis [Drosophila and Arabidopsis, it is unreliable to reach a conclusion on the RT model based on PP analysis.Using the abundance of PPs to test the RT model has limitations. Mammalian genomes have an especially high content of PPs and abundance of retrotransposons. In some organisms, transposable elements are subject to constant turnover . If PPs ublished ,64. Nonebidopsis ,24,27. CThe RT model, the genomic deletion model and the double-strand-break repair model attempt to describe how intron loss occurs. In evolution, the fate of a new mutation may be fixed, eliminated or randomly lost depending on its effect on the fitness of the host organism and the population size of the organism. Except that intron losses are neutral and thus randomly lost or fixed, the mutational models of intron loss cannot fully account for the pattern of intron loss. The inaccuracy of the mutational models to account for the patterns of intron loss in many studies may also be explained by the selective fixation of some special cases of intron loss -67. MiceReverse transcription is proposed as the main mechanism of intron loss in many species, and PPs are byproducts of RT. Therefore, if RT accounts for most of the intron-loss events, genes undergoing intron loss would produce more PPs, as observed in our study. This novel evidence of RT-mediated intron loss is consistent with previously proposed evidence, such as adjacent intron loss, 3'-biased intron loss and germline-biased expression of IL genes. We also found that both IL genes and parental genes of PPs are more likely to be translated on free cytoplasmic ribosomes where the LINEs are reverse transcribed. This phenomenon indicates that IL genes and parental genes of PPs are closely linked via their same subcellular translation locations. By contrast, in several imprecise IL genes whose introns were unlikely lost via RT process, no related PPs were found. The correlation between PP abundance and intron loss frequency more directly supports the RT model of intron loss than previously reported evidence. It provides a new strategy to test the model in eukaryotes that are rich in PPs.Mus musculus), rat (Rattus norvegicus) and six outgroup species, the guinea pig (Cavia porcellus), European rabbit (Oryctolagus cuniculus), human (Homo sapiens), cattle (Bos taurus), African bush elephant (Loxodonta africana) and gray short-tailed opossum (Monodelphis domestica) from Ensembl (Release 65) [Cricetulus griseus), from National Center for Biotechnology Information (Build 1.1) [C. griseus and C. porcellus, were selected because of their close relatedness with mice and rats whereas the other species were selected as outgroups because of their relatively large genome sizes among sequenced mammal genomes. A larger genome is likely to retain more orthologous genes and introns, which are essential for the identification of intron losses in mice and rats. Genes with obvious annotation errors such as those having coding sequences with non-multiples of three nucleotides or conflicting with their protein sequences were discarded. For genes with alternative splicing isoforms, we retained the longest mRNA for analysis.We downloaded the genome sequences and gene annotations of mouse (ease 65) , and anoild 1.1) . Two rodhttp://Pseudogene.org (Build 60 for mice and build 50 for rats) [Pseudogene annotations for mice and rats were obtained from or rats) ,71. Onlyor rats)  to conveE values < 10-10 and identities > 0.35. Where a protein from one species matched multiple proteins from other species , the genomic context was used to improve orthology assignment. OrthoCluster 2.0 [The best reciprocal BLAST was used to search orthologous proteins between mice and rats, with thresholds of ster 2.0  was usedClustal W 2.0  was usedC. griseus, C. porcellus, O. cuniculus, H. sapiens, B. taurus, L. africana and M. domestica were used to distinguish intron losses from intron gains  Percentage of mouse and rat genes producing processed pseudogenes. (B) Comparison of the abundance of processed pseudogenes and mRNA lengths between intron-lost genes and no-intron-lost genes in mice. (C) Comparison of lost introns and mRNA lengths between parents of processed pseudogenes and other genes in mice.Click here for fileImprecise intron loss events mediated by genomic deletion. (A) Sequence alignments around seven lost introns.  Another case of intron loss that may be mis-recognized as imprecise deletion.Click here for fileComparison of expression levels between intron-lost genes and no-intron-lost genes.Click here for filehttp://pseudogene.orgProcessed pseudogenes obtained from .Click here for file16,241 intron-containing orthologous genes between mice and rats.Click here for fileDetails for lost introns.Click here for fileList of intron-lost genes and no-intron-lost genes in mice and rats.Click here for file


Sarcoidosis is a multisystem inflammatory disease of unknown etiology affecting multiple organs. Earlier reports suggested that sarcoidosis was a disease of the developed world. However, recent reports suggest that the disease is found in the developing countries as well. Clinical, radiological, and histopathological similarities with tuberculosis pose a great challenge in countries endemic for tuberculosis. Mantoux test, high resolution computed tomography, and transbronchial lymph node and lung biopsies are diagnostic modalities, which play an important role in the diagnosis of sarcoid. In this review, we look at the epidemiology of sarcoid in tuberculosis-endemic regions, the sarcoidosis-tuberculosis link, clinical profile, diagnostic modalities, dilemma in the diagnosis, and the treatment of this disease. Sarcoidosis is a chronic, multisystem inflammatory disease of unknown etiology characterized by the presence of non-caseating granulomas in different organs. Although the lungs are the most common sites of inflammation, sarcoidosis can involve other organs such as the eyes , skin, lymph nodes, salivary glands, heart, spleen, liver, and the nervous system. A combiSarcoidosis was considered to be a disease seen more commonly in the west and rarely in the developing world in earlier reports. In a study from eight countries of Asia and Africa in 1976, fewer than 30 patients were reported from India, Malaysia, Thailand, Taiwan, and United Arab Emirates, while none from Singapore and Korea. In the The true burden of sarcoidosis in India is not clearly known, as reliable epidemiological data are not available. It has been estimated that sarcoidosis constituted 10 to 12 cases per 1,000 new registrations in a respiratory unit at Kolkata and 61.2/100,000 new cases at a center in New Delhi in a report published in 2002. The casMTb catalase peroxidase (mKatG), superoxide dismutase, mycolyl transferase, and heat shock proteins have been detected[Mycobacterium tuberculosis as a cause of sarcoidosis has been extensively studied in the literature-25. Repodetected; immune detected-33. Thisdetected.The course of sarcoidosis ranges from asymptomatic to severe disease. Male preponderance with a mean age of 40 years is reported in most Indian reports,4,7,10. The disease affects predominantly the lungs, thoracic lymph nodes, skin, and eyes. Clinically, patients may be asymptomatic or complain of cough or breathlessness. Constitutional symptoms such as fever, malaise, fatigue, and weight loss may be present. The most frequent intrathoracic involvement is the presence of hilar or mediastinal lymph node enlargements with or without radiologic evidence of interstitial involvement in one-third of patients. The miliary pattern can be seen in sarcoidosis and may be misdiagnosed and treated for tuberculosis. High reSkin involvement may include sarcoid granulomas and erythema nodosum. Bilateral parotid gland enlargement, hepatic involvement with granulomas, renal insufficiency, myocardial involvement with heart failure, hypertension and conduction abnormalities, arthropathy, neurological involvement in the form of recurrent laryngeal nerve palsy, Bells' palsy, other cranial neuropathies and spinal chord involvement, generalized lymphadenopathy, and minor hematologic abnormalities have been reported from India,4. SporaPatients may present directly to an ophthalmology clinic with blurred vision, floaters, redness or discomfort, or may be referred by the pulmonologists or rheumatologists for an ocular evaluation. Ocular disease may be the initial manifestation in sarcoidosis. Ocular involvement occurs in 25% to 60% of patients with systemic sarcoidosis presenting to a respiratory center. This waIn contrast to reports from western literature, panuveiAnterior uveitis could be acute iridocyclitis or a chronic granulomatous uveitis with keratic precipitates, which may vary from cellular to large mutton fat type of keratic precipitates. Iris nodules such as the Koeppe and busacca nodules and iris granulomas may be seen. Granulomas may also be seen in the trabecular meshwork causing increase in intraocular pressure at the time of activity. Intermediate uveitis with vitritis, peripheral vasculitis, snowball opacities, and snow banking have been described in reports from India as well. Posterior segment involvement includes periphlebitis with characteristic ‘candle wax dripping appearances’ , multifocal chorioretinitis, peripheral punched out chorioretinal lesions, choroid and optic nerve granulomas, optic disc edema, papilledema, optociliary shunts, and arterial macro aneurysms. Rare manifestations of scleral nodule, vasoproliferative tumors, and retinitis have also been reported from India. Conjunctival granulomas, bilateral lacrimal gland enlargement, and orbital mass lesions have been reported from India,34. IlluA negative Mantoux (PPD) skin test is considered to suggest sarcoidosis and is usually seen in 90% of sarcoid patients. ElevateHigh resolution CT is of great help in making the diagnosis of sarcoidosis by detecting interstitial involvement and lymphadenopathy. Contrast-enhanced CT may help distinguish sarcoid from tubercular lymph node enlargement, which would usually show a central hypodensity corresponding to the caseation and peripheral contrast enhancement. The frequent use of fiberoptic bronchoscopy with transbronchial lymph node and endobronchial lung biopsies with diagnostic yield of about 80% have helped in the recognition of sarcoidosis. Microbiology (smear and culture for MTB) and histopathology are routinely done on the aspirates/biopsy material to rule out TB.Both TB and sarcoid share remarkable similarities in their clinical and radiological presentations. Both diseases present with constitutional symptoms of fever, malaise, weight loss, and fatigue. Respiratory symptoms are common to both diseases. Similar ocular manifestations are seen in both diseases. Serpiginous-like choroiditis in the Indian subcontinent are more likely to be associated with tuberculosis; howeverRadiologic similarities may be seen in both diseases. Discrete, bilateral, symmetrical lymph node enlargement is usually seen in sarcoidosis. Fibrosis and military distribution although common in TB may be seen in both diseases. Both diseases show granulomatous inflammation on histopathology. Caseation is seen in tuberculosis. Fibrinoid necrosis though rare can occur in up to 30% of cases of sarcoidosis. There are no specific features enough to differentiate the two conditions with certainty, except a positive culture of MTb. A positive PCR for MTb may be seen in the biopsy samples of sarcoidosis.Differentiating the two conditions pose a challenge, particularly in countries with high prevalence of TB. A combination of clinical, radiologic, and laboratory tests are used to make a diagnosis of sarcoidosis. Tuberculin skin test plays an important role in the diagnosis. TubercuCorticosteroids are the mainstay of treatment in sarcoidosis. In chronic cases or in those patients were steroids are contraindicated, immunosuppressants like methotrexate, azathioprine, mycophenolate mofetil, cyclosporine, and cyclophosphamide have been used,40. MethDiagnosing sarcoid in countries with high TB burden does pose a significant challenge. However, new cases of sarcoid are increasingly diagnosed in TB endemic areas in recent years due to increased awareness and better availability of diagnostic modalities. Ocular evaluations do contribute in making a diagnosis of systemic sarcoid. Negative Mantoux test is still being considered an important diagnostic test to make a diagnosis. Sarcoidosis does exist in populations with high TB burden, and the association between sarcoid and TB continues to be an enigma. Prospective studies from the Indian continent on the epidemiological data, genotypic and phenotypic variations, and the role of mycobacteria in the development of sarcoidosis would contribute a great deal in understanding this disease.Written informed consent was obtained from the patient for the publication of this report and any accompanying images. The consent to publish has been provided by the patient with the face photograph and has been approved by our Institutional Review Board .The author declares that she has no competing interests.