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The majority of the genome is transcribed into pieces of non-(protein) coding RNA, among which long non-coding RNAs (lncRNAs) constitute a large group of particularly versatile molecules that govern basic cellular processes including transcription, splicing, RNA stability, and translation. The frequent deregulation of numerous lncRNAs in cancer is known to contribute to virtually all hallmarks of cancer. The post-transcriptional regulation of lncRNAs is mediated by RNA-binding proteins (RBPs). Interestingly, RBPs themselves are commonly deregulated in cancer and could thus constitute a major contribution to the deregulation of cancer-associated lncRNAs. Discussed here are four examples of well-known RBPs that regulate the transport or localization of cancer-associated lncRNAs and thereby impact the functionality of these lncRNAs. So far, out of the vast number of RBPs that exist, only a relatively small number has been found to specifically guide the transport or localization of cancer-related lncRNAs. In general, there is still a lack of knowledge about how lncRNAs are shuttled between or retained within different cellular compartments and future research will have to shed more light on these regulatory mechanisms.
The concept that RNA merely serves as an intermediate, conveying the genetic information encoded in the form of DNA to be translated into proteins, has long been overthrown . In fact, the vast majority of the genome does not code for proteins but is transcribed into various types of so-called non-coding RNAs (ncRNAs), which by themselves fulfill a multitude of pivotal regulatory functions . Among these ncRNAs, one large group with particularly versatile functions are the long non-coding RNAs (lncRNAs), a class of ncRNAs that are defined as being longer than 200 nucleotides . These lncRNAs can originate from different genomic locations. Most are interspersed between protein-coding genes (long intergenic non-coding RNAs, lincRNAs), while others are transcribed from the sense or antisense strands of introns and also exons of coding genes, and yet another type of lncRNAs originates from enhancer regions (eRNA) .
Compared to protein-coding genes, lncRNAs are poorly conserved between different species and their expression levels are rather low . Initially, this led to the belief that they were nothing but transcriptional noise . Soon, however, it was discovered that lncRNAs do exhibit considerable functionality, for example as regulators of transcription, but also on the post-transcriptional level by regulating mRNA splicing, stability and translation . LncRNAs can also act as so-called competitive endogenous RNAs (ceRNAs), which sponge up microRNAs (miRNAs) and thus prevent them from binding to and inducing degradation or translational repression of their mRNA targets .
For a lncRNA to fulfill its function it is not only important how much of this lncRNA is present in a cell but equally, or potentially even more important, whether the lncRNA is properly transported to and located at its site of action. While lncRNAs are frequently enriched in the nucleus, they can also be located in the cytoplasm or in mitochondria and can be shuttled between these different compartments in response to different cellular conditions . The transport of lncRNAs constitutes a form of post-transcriptional regulation that is mediated by RNA-binding proteins (RBPs) . There is, however, not a lot known about how this transport of lncRNAs is facilitated . Discussed below are four examples of well-known RBPs that have been found to regulate the transport or localization of cancer-associated lncRNAs. Importantly, these RBPs are themselves commonly deregulated in cancer and thus potentially contribute to the deregulation of lncRNAs in cancer.
HuR, the protein product of the ELAV1 gene, is a ubiquitously expressed RBP that contains three RNA recognition motifs (RRMs) via which it preferentially binds to adenylate/uridylate-rich RNA elements (AREs) . AREs are signals for rapid RNA degradation, and by blocking these recognition sites HuR can stabilize its RNA interaction partners . HuR is frequently upregulated in cancer cells and is known to be involved in many hallmarks of cancer, such as invasion, angiogenesis, and inflammation, by post-transcriptionally regulating various cancer-related mRNAs . In addition, HuR is known to not only regulate the level of lncRNAs by affecting their stability but has also been found to have an impact on lncRNA localization . The different regulatory mechanisms that HuR exerts on its lncRNA targets are illustrated in Figure 1.
Figure 1. The RNA-binding protein (RBP) human antigen R (HuR) can regulate long non-coding RNAs (lncRNAs) by different means. First, by binding to its target lncRNA in the nucleus it can facilitate its subsequent nuclear export by interaction with the importin β superfamily member chromosomal maintenance 1 (CRM1). Secondly, by shielding lncRNAs from RNA decay promoting RBPs like ARE/poly(U)-binding/degradation factor 1 (AUF1) and tristetraprolin (TTP), HuR can enhance lncRNA stability. Thirdly, by recruitment of let7–Argonaute-2 (Ago2) it can promote lncRNA degradation via the RNA-induced silencing complex (RISC).
A study by Noh et al. found HuR to bind to both the 3′ and 5′ end of the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a 265-nt-long lncRNA that is best known as a component of the mitochondrial RNA-processing endoribonuclease (RNase MRP) complex and that is also involved in mitochondrial DNA replication . Rather recently, an additional role of RMRP in different types of cancer has been reported by multiple publications that describe a contribution of RMRP to, for example, cancer cell proliferation, migration, and invasion, by sponging up different miRNAs . According to Noh and colleagues, HuR binds to RMRP already in the nucleus and subsequently facilitates its nuclear export . HuR itself can shuttle back and forth between the nucleus and cytoplasm in response to different stimuli . The export of HuR from the nucleus occurs in a chromosomal maintenance 1 (CRM1)-dependent manner . CRM1 is a member of the importin β superfamily that facilitates the nuclear export of proteins, to which it can bind either directly or via adaptors, as well as of RNA, in which case adaptor proteins are necessary . Noh et al. showed that not only silencing of HuR itself in HEK293 cells but also of CRM1 led to significantly reduced cytoplasmic levels of nascent RMRP . Importantly, there was no additive effect when both HuR and CRM1 were knocked down, highlighting that the nuclear export of RMRP was facilitated via the HuR–CRM1 axis . While affecting the localization of RMRP, HuR was not observed to have an impact on the steady-state levels, meaning the stability, of the lncRNA . There are a number of cancer-related lncRNAs whose stability is regulated by HuR, for example NEAT1 and lncRNA-p21  and based on the high frequency of HuR binding sites, particularly in intronic regions , it can be anticipated that more will be identified in the future. However, so far, RMRP is the only lncRNA whose nuclear export has been reported to be mediated by HuR.
Even though the lncRNA RMRP is encoded in the nucleus, it is also present in mitochondria . As detailed above, Noh et al. uncovered that the first step in the transport pathway of RMRP, the export from the nucleus into the cytoplasm, is facilitated by the RBP HuR . In the same study the authors also reported that the subsequent import of RMRP into mitochondria seemed to be mediated via two import machineries, the TOM/TIM machinery and polynucleotide phosphorylase (PNPase) . The TOM/TIM machinery consists of a translocase complex of the outer mitochondrial membrane (TOM) and two different inner membrane translocases (TIM) that together enable the transport of proteins across both mitochondrial membranes into the matrix . The PNPase is located in the mitochondrial intermembrane space and promotes the import of RNA from the cytoplasm into the mitochondrial matrix . How exactly RMRP is transported by these machineries and which RBPs it interacts with in order to do so has not been elucidated . What Noh et al. did report was that the RBP GRSF1, while not directly contributing to its import, promoted the accumulation of RMRP in the mitochondrial matrix . GRSF1 contains three RRMs via which it binds to a G-rich recognition motif (AGGGGD, with D = A/U), subsequently regulating splicing, polyadenylation, and export of its RNA targets . One isoform of GRSF1 is located in mitochondria where it forms granules with newly synthesized mitochondrial RNAs, among them the two mitochondrial lncRNAs lncCyt b and lncND5, which carry 10 and 21 GRSF1 consensus-binding sites, respectively . Knockdown of GRSF1 in immortalized primary fibroblasts decreased the overall level of these two mitochondrial lncRNAs by about half . Noh et al., however, reported that the whole-cell level of RMRP was not affected by GRSF1 knockdown, but only its accumulation in the mitochondrial matrix . Hence, GRSF1 seems to retain RMRP in the matrix once it has been imported, thereby facilitating its enrichment at its specific site of action .
RMRP is not the only example of a nuclear-encoded lncRNA that can be transported into mitochondria. The cancer-associated lncRNA MALAT1 (Metastasis-associated lung adenocarcinoma transcript 1) was also found to be present in mitochondria . Under normal conditions, MALAT1 is mostly located in the nucleus where it regulates alternative splicing and gene expression . In hepatocellular carcinoma (HCC), however, Zhao et al. observed an increased level of MALAT1 within mitochondria . An RNA-fluorescent in situ hybridization (FISH) assay combined with mitochondrial staining showed that MALAT1 was highly enriched in HepG2 mitochondria but barely detectable in non-cancerous hepatic cells . This mitochondrial accumulation of the lncRNA in HCC appears to enhance mitochondrial energy metabolism, thus potentially contributing to the oncogenic effects that MALAT1 exerts in HCC . Interestingly, the study observed that for the mitochondrial lncRNA lncCyt b the situation is the other way around, namely that in non-cancerous cells it is located primarily in the mitochondrial matrix, where it is bound by GRSF1, but in HepG2 cells, it shows increased presence in the nucleus . How the shuttling of these two lncRNAs between the nucleus and mitochondria is mediated is not known so far. It is clear, however, that different RBPs, like HuR and GRSF1, must play a central role in this process and that deregulations in this network of RBPs are responsible for the aberrant localization of lncRNAs in cancer, as observed for MALAT1 and lncCyt b in HCC. In case of lncCyt b, a disturbed interaction with GRSF1 could be one factor contributing to this phenomenon.
IGF2BP1, together with IGF2BP2 and IGF2BP3, belongs to a family of RBPs that is expressed in embryonic tissue and frequently reactivated in different types of cancer but rarely expressed in normal adult tissue . Due to its regulation of stability, localization, and translation of numerous mRNAs like c-Myc , beta-catenin , and KRAS , it plays a role in embryogenesis and tumor development . IGF2BP1 carries two RRMs in its N-terminal region and four hnRNP-K homology (KH) domains in the C-terminus, which are primarily responsible for its interaction with RNA targets in an N6-methyladenosine (m6A)-dependent manner . The m6A modification is the most common type of RNA modification both in mRNAs and lncRNAs and stems from the addition of a methyl group to the N-6 position of adenosine by proteins like methyltransferase-like 3 (METTL3), METTL14, and Wilms’ tumor 1-associating protein (WTAP) .
IGF2BP1 is mostly cytoplasmic where it forms, together with its RNA targets and other RBPs, so-called messenger ribonucleoprotein (mRNP) granules . When not associated with these mRNPs, IGF2BP1 can also translocate to the nucleus where it has been found to bind to mRNA already during transcription . As IGF2BP1 contains two nuclear export signals (NES) within its second and fourth RNA-binding KH domain it is subsequently exported back into the cytoplasm together with its bound RNA target . Hence, IGF2BP1 facilitates the nuclear export of its RNA target . The prime example of this is given by beta-actin. IGF2BP1 binds to the beta-actin mRNA as soon as it is transcribed and subsequently enables its export into the cytoplasm, where IGF2BP2 either locates to perinuclear regions or interacts with and moves along the cytoskeleton towards the cell periphery, more precisely towards newly forming lamellipodia . Here, IGF2BP2 is phosphorylated by the Src-kinase, which results in the disassociation of the beta-actin mRNA and its localized translation .
Following the same mechanism as observed for the beta-actin mRNA, IGF2BP1 also regulates the subcytoplasmic distribution of the lncRNA H19 . H19 is a 2.3-kb-long, spliced and polyadenylated lncRNA that is, similarly to IGF2BP1, expressed during embryonic development and reactivated in several types of cancer . H19 shows a diverse range of actions: it functions as a ceRNA, induces or represses the transcription of various genes, and interacts with and thus modulates the activity of different proteins like p53, thereby contributing to all hallmarks of cancer . Runge et al. discovered that IGF2BP1 binds to the 3′ end of H19 with high affinity and thereby targets the lncRNA to lamellipodia and perinuclear regions of proliferating mouse embryonic fibroblasts . In growth-arrested confluent cells, the IGF2BP1–H19 complex was dispersed more evenly in the cytoplasm . It has been discovered that H19 seems to contribute to the migratory behavior and branching morphogenesis of epithelial cells, processes that are essential during embryogenic development but that also enable migration, invasion, and metastasis of cancerous cells . This function of H19 coincides well with the targeting of IGF2BP1-H19 to the leading edge of proliferative cells.
The above discussed examples of RBPs are involved in the transport or localization of cancer-associated lncRNAs outside of the nucleus. In general, lncRNAs, however, tend to be enriched in nuclear fractions . A well-studied RBP that plays a role in the nuclear accumulation of lncRNAs is the heterogeneous nuclear ribonucleoprotein K (hnRNPK) . The exact mechanism of how hnRNPK can retain RNAs in the nucleus is however still unknown . HnRNPK fulfills a wide variety of cellular functions, for example by acting as a transcription factor, regulating translation, and serving as a hub for various signaling pathways . Numerous studies have observed oncogenic effects as well as a prognostic relevance of hnRNPK in different types of cancer, like breast, colorectal, and gastric cancer, where its overall levels are increased and it is aberrantly localized in the cytoplasm . The binding of hnRNPK to RNA occurs via the interaction of its three KH domains, which are a type of RNA-binding motif first discovered in and therefore named after the RBP, with poly-C sequences in the RNA targets . Lubelsky and Ulitsky, by screening a library of short fragments from nuclear mRNAs and lncRNAs, discovered a specific consensus sequence that is bound by hnRNPK and mediates nuclear accumulation . This sequence consists of a 42-nt-long fragment that overlaps with Alu repeats, a very common type of short interspersed element (SINE), in antisense orientation and that contains three stretches of at least six pyrimidines (C/T), with two of these stretches matching the consensus sequence RCCTCCC (R = A/G) . They termed the sequence SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) .
SIRLOINs are found in 13.1% of human lncRNAs and 7.5% of mRNAs and contribute substantially to the nuclear enrichment of RNA transcripts . An example of a cancer-associated lncRNA that contains a SIRLOIN and is retained in the nucleus by hnRNPK is MALAT1 . As stated before, the nuclear accumulation of MALAT1 has, however, been found to be disturbed in HCC, where undefined reasons lead to an increased mitochondrial presence of the lncRNA . A recent study by Nguyen et al. showed that the deletion of a SINE in MALAT1 and the hence disrupted interaction with hnRNPK resulted in a more frequent translocation to the cytoplasm . This was accompanied by increased DNA damage and apoptosis due to the redistribution of a protein called transactive response DNA binding protein 43 kDa (TDP-43) to the cytoplasm along with MALAT1 to which it is bound . Aberrant expression and localization of hnRNPK, commonly observed in cancer, could thus also play a role in the change of MALAT1 localization in HCC. As reviewed elsewhere, there are numerous examples for interactions between lncRNAs and hnRNPK where they jointly regulate gene expression by various means . Thus, the deregulation of hnRNPK in cancer has far-reaching ramifications as it affects the function and localization of lncRNAs .