Paraspeckles

Paraspeckles were discovered in 2002 by Andersen and colleagues during the study of the proteome of purified nucleoli of human cells using mass spectrometry . During the study, 271 proteins were isolated, of which 30% were previously unknown. A more detailed study of one of these newly discovered proteins showed that it does not accumulate in the nucleoli at all, but diffuses in the nucleoplasm , but is highly concentrated in 5–20 nuclear foci. It turned out that these tricks do not overlap with any of the previously known nuclear bodies, and they were called “paraspeckles” due to the location of speckles near other nuclear bodies. The protein itself was called PSPC1 (from the English. Paraspeckle Protein 1 ) ^[1] .

Paraspeckles are small bodies of irregular shape. Depending on the type of cells, usually in the nucleus there are from 5 to 20 para-speckles. A study using electron and fluorescence microscopy showed that human paraspecules have a width of about 360 nm (a little less in the mouse) and a length of 1 to 2 microns . Reliably the existence of paraspeckles is shown only for mammalian cells. In humans, paraspeckles are absent in embryonic stem cells and induced pluripotent stem cells . Orthologs of key paraspeckle proteins are found in other vertebrate and invertebrate animals , however, the long non-coding NEAT1 RNA necessary for paraspeckle formation is found only in mammals, which, apparently, is associated with the absence of paraspeckles in the nuclei of other organisms. The number and length of paraspeckles are proportional to the level of expression of the long NEAT1 isoform ^{[3] [2]} .

Recent studies using electron microscopy and have shown that paraspeckles are chains of fused spherical subcompartments, each of which has a crustal part and a shell. The 5'- and 3'-ends of the long NEAT1 isoform are localized in the shell, and the rest of the molecule is in the core. Different proteins of paraspeckles are also localized in different parts of paraspeckles ^[2] .

Paraspeckles are located in the interchromatin space, sandwiched between the larger nuclear speckles and chromatin . It was shown that, inside the paraspeckles, RNA polymerase II is inactive, and transcription mediated by this enzyme occurs only at the edge of the paraspecs ^[4] .

The relationship of paraspeckles with the nucleolus is not yet fully understood, however, one of the key proteins of paraspeckles, PSPC1, can move between paraspecks and the nucleolus. In addition, while suppressing the work of RNA polymerase II, paraspeckle proteins are localized in special perinucleolar caps ^[4] .

More than 40 different proteins ^[2] and RNA accumulate in large quantities in paraspeckles. Most paraspeckle proteins are associated with transcription mediated by RNA polymerase II and RNA processing . One of the two RNAs that accumulate in paraspeckles is called Ctn and is involved in the regulation of gene expression by retention of RNA in the nucleus. The second RNA is called NEAT1 and plays an architectural role, being necessary for the formation and maintenance of the paraspectrum structure. The main components of paraspeckles are listed in the table below ^[5] .

Squirrels

The most important components of paraspeckles are three proteins of the DBHS family: PSF / SFPQ, NONO / P54NRB and PSPC1. They are localized in the nucleoplasm and in the para speckles. Most often, PSPC1 protein is used as a marker for paraspeckles, since it accumulates to a lesser extent in the nucleoplasm. The two N-terminal RNA-binding motifs and the C-terminal these proteins are 50% identical in sequence. These proteins play an important structural role in para speckles: for example, knockdown of the P54NRB and PSF proteins in HeLa cells leads to the loss of para speckles. Compared to P54NRB and PSF, PSPC1 is not so abundantly expressed, and its knockdown does not lead to the loss of paraspeckles. These three proteins interact with each other and, most likely, inside the cells they are in the form of homo- or heterodimers . Interaction with each other is carried out through the bispiral motive. To localize these proteins in paraspeckles, both domains containing C-terminal bispiral motifs and domains are required. Members of the DBHS family bind single and double stranded DNA and RNA molecules and participate in many stages of RNA synthesis and processing. In addition, PSF and P54NRB bind and retain transcripts inside the nucleus that underwent intensive editing of adenosine → inosine. They also function in the cytoplasm , for example, PSF and P54NRB are part of RNA transfer granules in dendrites , however, only P54NRB can be actively transported from the nucleus to the cytoplasm. Interestingly, P54NRB is involved in the regulation of circadian rhythms in mammals ^[6] .

Paraspeckle proteins are considered to be those proteins that are colocalized at specific nuclear loci together with proteins of the DBHS family, however, the overall picture of the localization of these proteins can vary significantly. For example, CFIM68 protein can be observed in para speckles, but it also occurs in nuclear speckles; at the same time, RNA polymerase II, in addition to paraspeckles, is detected in chromatin and nuclear speckles. Finally, some proteins are found in para speckles only under overexpression conditions, for example, BCL11A, WTX, WT1 (+ KTS) and CoAA ^[7] .

Paraspeckl proteins that are not members of the DBHS family are usually transcription factors or transcriptional regulators. CoAA functions as a transcriptional coactivator that regulates mediated transcription and alternative splicing . WTX is a transcriptional regulator and tumor suppressor; it coactivates the transcription of WT1 protein, which can also be localized in paraspeckles. SOX9 is a transcription factor that plays a critical role in bone formation. It directly interacts with P54NRB, and its overexpression leads to a change in the localization of P54NRB and PSPC1. BCL11A is a transcription factor that is involved in the development of / leukemia. Cutting factor CFIM68 is necessary for the first stage of processing the 3'-end of pre-mRNA ^[8] .

For the formation of paraspeckles, NONO and SFPQ proteins are strictly required. In addition, knockdown of proteins such as HNRNPK, DAZAP1, FUS, RBM14 and HNRNPH3 leads to the absence of paraspeckles ^[2] .

RNA

Prior to describing the specific RNAs that make up the para speckles, there was some evidence that para speckles do contain RNA in addition to proteins. First, paraspeckles are destroyed by processing the cells with RNase . Secondly, all the most important paraspectrum proteins contain motifs that bind RNAs; moreover, all these proteins participate in one way or another in RNA processing. Thirdly, for the localization of PSPC1 in paraspeckles, the RNA-binding domain of this protein is required. Finally, paraspeckles disappear upon suppression of transcription mediated by RNA polymerase II, and reappear when transcription is resumed. Two RNAs that are part of the paraspectrum are known : Ctn and NEAT1 / Men ε / β ^[9] .

Ctn was the first paraspectral RNA discovered; it was described in 2005. This is a mouse-specific poly (A) tail transcript that is read from the mCAT2 locus. Ctn contains all exons of the CAT2 transporter protein, however, unlike the mRNA of this protein, Ctn is synthesized from another promoter and has a longer 3'-untranslated region (3'-UTR). Ctn is localized not only in para speckles , but also in the nucleoplasm. The 3'-UTR of this RNA undergoes intensive editing A → I, and inverted repeats are edited, due to which double-stranded regions are formed in the RNA structure. Paraspeckle protein P54NRB binds to inosine in RNA, which, in addition, binds to Ctn in vivo . Probably, due to the binding to proteins, the Ctn paraspeckles remain in the para-speckles and are not exported to the cytoplasm. Under the action of a number of stress signals, the 3'-NTO Ctn is cut, and the amount of this RNA in the nucleus decreases ^[10] .

In 2009, several research groups independently discovered the second paraspeckle RNA, NEAT1 . This long non-coding RNA is necessary for the formation and maintenance of the structural integrity of paraspeckles. Two variants of NEAT1 are known , differing in their length; the structural role in paraspeckles is played by the long isoform ^[2] . Interestingly, the long NEAT1 isoform is cut very close to the 3'-end, which forms a very short RNA molecule, similar to tRNA . Knockdown of NEAT1 leads to a complete loss of paraspeckles, and overexpression of this RNA leads to an increase in the number of paraspeckles in some . In addition, para speckles form near the NEAT1 genes. Apparently, as in the case of Ctn , the localization of NEAT1 in para speckles is associated with interaction with proteins of the DBHS family, although NEAT1 does not undergo editing A → I. By changing the amount of NEAT1 in the cell, one can judge the functioning of para speckles . Human embryonic stem cells, as well as induced pluripotent stem cells ^[2] do not express NEAT1 and do not have paraspeckles, despite the expression of DBHS proteins, however, paraspecules appear when differentiation begins. In addition, with the advent of paraspeckles, the accumulation in the nucleus of mRNAs subjected to editing A → I is enhanced ^[11] .

According to the existing model, the formation of para speckles begins with the formation of NEAT1 transcripts in the nuclei of daughter cells shortly after cell division. Shortly after synthesis, NEAT1 molecules form complexes with DBHS proteins, and NEAT1 transcripts do not have time to move away from their locus. The formed para speckles most likely consist of many NEAT1 complexes with DBHS proteins and are quite dynamic: individual molecules of DBHS proteins can enter the nucleoplasm and vice versa. It is likely that the ability of DBHS proteins to oligomerize, as well as the interaction directly between NEAT1 RNA molecules themselves, is involved in the formation of paraspeckles. In the absence of NEAT1, para speckles are unable to maintain their structural integrity and are not re-formed ^[12] . According to recent data, phase separation at the liquid – liquid interface is involved in the formation of para speckles ^[2] .

The main function of paraspeckles is the retention in the nucleus of mRNAs containing double-stranded regions formed by and edited by adenosine → inosine. Retention of RNA in the nucleus is associated with a cellular response to stress, viral infections , as well as maintaining circadian rhythms. Apparently, paraspeckles are involved in cell reprogramming, which occurs during differentiation; perhaps, by retaining RNA in the nucleus, paraspecules alter the expression of key proteins. The absence of NEAT1 and paraspectrum could potentially serve as a marker of pluripotency . Paraspectrum protein CFIM68 can regulate the release of RNA from the nucleus by introducing sections into RNA ^[13] .

Paraspeckles can selectively accumulate within themselves certain proteins, leading to a decrease in the concentration of these proteins in other places. This, in turn, can affect the expression of a number of genes ^[2] .

According to the latest data, paraspeckles can be localized directly at the sites of the beginning of transcription of actively transcribed genes. Proteins of paraspeckles SFPQ and NONO are involved in the processing of miRNAs , and the accumulation of these proteins in paraspecks contributes to the efficient processing of miRNAs ^[2] .

Paraspeckles and NEAT1 are not strictly necessary for the development of mammals under normal conditions, since mice lacking NEAT1 are viable. However, some females of such mice showed deviations in the formation and functioning of the ovarian secretory glands and the corpus luteum , which reduces fertility . Possibly, paraspeckles perform some evolutionarily conservative function in the cells of the secretory structures of the female reproductive system of mammals: for example, the possum has well-formed paraspecs in the cells of the uterine glands. It is curious that similar effects were observed not in all knockout females, but only in some, which can be associated with the need for paraspeckles to function under stressful environmental conditions ^[2] .

The expression level of the long NEAT1 isoform increases when cells are infected with certain , such as Japanese encephalitis virus, rabies , HIV , influenza virus and Huntaan virus , as well as herpes simplex DNA virus . In most cases, this increase is a protective mechanism. An increase in the number of paraspeckles leads to the accumulation of a large number of proteins in them, which the virus cannot use for its reproduction, as well as a change in the expression of a number of genes ^[2] .

In the case of cancer, NEAT1 expression may decrease, increase compared to normal tissue, or remain unchanged. Mutations in the NEAT1 gene are associated with the development of liver cancer and breast cancer . There is evidence that NEAT1 may even stimulate the formation of metastases ^[14] . An increase in NEAT1 expression and an increase in the number of paraspeckles may be due to the stressful conditions in which the cancer cells are located, but paraspeckles may also have an oncogenic role. In the case of prostate cancer, an increase in the number of paraspeckles and expression of NEAT1 corresponds to more aggressive forms of the disease. However, there is evidence that paraspeckles can suppress the development of tumors ^[2] .

Paraspeckles can play special roles in the functioning of the nervous system. It was shown that after an epileptic seizure, expression of the long NEAT1 isoform increased in some parts of the mouse brain . Paraspeckles can be involved in the development of amyotrophic lateral sclerosis ^[2] .

There is evidence that NEAT1 can regulate the inflammatory process by triggering the formation of para speckles in macrophages ^[15] .

• Razin S.V., Gavrilov A.A. Organization of functional processes in the cell nucleus: an order arising from disorder // Moscow University Bulletin. Ser. 16. Biology. - 2015. - No. 3 . - S. 13-20 .
• The Nucleus / Tom Misteli, David L. Spector. - N. Y .: Cold Spring Harbor Perspectives in Biology, 2011 .-- 463 p. - ISBN 978-0-87969-894-2 .