(A) The MALAT1 locus, located on mouse chromosome 19, has been reported to yield a ~7-kb non-coding transcript (transcription start site denoted by arrow) that contains regions of high conservation. MALAT1 lacks repetitive elements except for a SINE and LINE element near its 5’ end. (B) Small RNAs were isolated from EpH4-EV (denoted EV) and EpH4-A6 (denoted A6) cells. Four different probes that span the mMALAT1 locus were used for Northern blot analysis as designated at the bottom of each blot. U6 snRNA was used as a loading control. (C) Strand-specific oligonucleotide probes were used to further map the 61-nt small RNA. Sense probes detect transcripts originating from the opposite strand of mMALAT1, while antisense probes detect transcripts originating from the same strand as mMALAT1. (D) The 61-nt mascRNA transcript is highly conserved (Sequence shown as DNA and does not include CCA at the 3’ end, which is added during processing). (E) The mascRNA transcript lacks translation initiation and stop codons.

(A) Northern blot analysis using strand-specific oligonucleotide probes showed that the human mascRNA ortholog is expressed in HeLa cells. U6 snRNA was used as a loading control. (B) 10 µg of total RNA from twenty normal human tissues were probed for mascRNA expression. (C) A 25-nt probe complementary to nt 6728–6752 of mMALAT1 was designed. The 25-nt probe specifically distinguished expression of the mouse isoform in EpH4-EV cells from the human isoform in HeLa cells.

(A) A 5’-phosphate-dependent exonuclease was used to show that mascRNA has a 5’-monophosphate group. In lanes 3–6, EpH4-A6 total RNA was first treated with antisense oligonucleotides (as designated at top) complementary to mascRNA and subjected to RNase H treatment. A probe complementary to nt 6690–6709 of mMALAT1 was used for the Northern blot. The microRNA let-7a was used as a positive control for exonuclease activity, whereas U4 snRNA is capped and serves as a loading control. (B) The mature mouse mascRNA transcript is predicted to form a tRNA-like cloverleaf secondary structure. The four point mutations between the mouse and human orthologs are shaded. The * designates the nucleotide that is commonly modified. (C) The 3’ end of mouse mascRNA was cloned using a ligation-based approach (3’ linker oligo is designated). All sequenced mascRNA cDNA clones end in CCA and six of eight clones show a common nucleotide modification. (D) Total RNA from HeLa cells treated with 50 µg/mL of α-amanitin, an RNA polymerase II inhibitor, for 6 or 12 hours was subjected to Northern blot analysis. (E) EpH4-EV and EpH4-A6 cells were fractionated to isolate nuclear and cytoplasmic total RNA, which was then subjected to Northern blot analysis using a probe to nt 6563–6843 of mMALAT1. The long mMALAT1 transcript was exclusively nuclear, whereas the mascRNA transcript was exclusively cytoplasmic.

(A) Antisense probes complementary to mMALAT1 were designed and used in Parts (B) and (C). (B) 10 µg of total RNA from EpH4-A6 cells was probed by Northern blot analysis using the designated oligonucleotide probes. β-actin was used as a loading control. (C) Northern blots revealed that the 3’ end of the abundant mMALAT1 transcript is further upstream than previously reported and, therefore, the abundant transcript is only ~6.7-kb in length. (D) A conserved poly(A)-rich tract is present immediately upstream of mascRNA. Numbers at the top denote the mouse nucleotide position, whereas numbers at the bottom denote the corresponding human nucleotide position. (E) RNase H digestion followed by Northern blot analysis was used to map the 3’ end of the abundant human MALAT1 transcript in HeLa cells. ASO1 is complementary to nt 7381–7430 of hMALAT1. ASO2 is complementary to nt 7331–7380 of hMALAT1. Both antisense oligonucleotides result in RNase H cleavage products that suggest the 3’ end of the long hMALAT1 transcript is at ~nt 7520. (F) Total and poly(A)⁺ RNA were isolated from EpH4-EV cells and probed by Northern blot analysis to show that the ~6.7-kb mMALAT1 transcript is present in the poly(A)⁺ fraction.

(A) Antisense oligonucleotides (ASOs) complementary to the mascRNA transcript and flanking regions were designed. Numbers to the left of each ASO represent lane number in Panel B. Probe A recognizes both the ~6.7-kb and ~7-kb mMALAT1 isoforms, whereas Probe B only recognizes the ~7-kb mMALAT1 isoform. (B) Northern blots were performed to determine the effect of ASO treatment on expression of the ~7-kb mMALAT1, ~6.7-kb mMALAT1, and mascRNA transcripts. The data in the bar graphs are shown as mean and standard deviation values of three independent transfections. Representative Northern blots are shown.

(A) After ASO transfection, EpH4-EV cells were fractionated to isolate nuclear and cytoplasmic total RNA. The ~7-kb mMALAT1 transcript was nuclear-retained and ASO treatment did not affect the cytoplasmic localization of mascRNA. U6 and let-7a were used as controls for fractionation efficiency. (B) MALAT1 can be cleaved in vitro by E. coli RNase P. M1 RNA from E. coli was incubated for 1 hr at 37 degrees with uniformly labeled MALAT1 substrates. Samples were then electrophoresed in a 8% polyacrylamide/8 M urea gel. (C) MALAT1 can be cleaved in vitro by partially purified human RNase P. (D) Recombinant His-tagged human RNase Z cleaves MALAT1 in vitro at the 3’ end of mascRNA.

Cleavage/polyadenylation can occur to yield a ~7-kb mMALAT1 transcript, but this represents a minor product that is expressed at a very low level in cells. MALAT1 is primarily processed via an upstream cleavage mechanism, which yields a mature ~6.7-kb mMALAT1 transcript with a short poly(A) tail-like moiety at its 3’ end. Endonucleolytic cleavage by RNase P simultaneously generates the 3’ end of the ~6.7-kb MALAT1 transcript and the 5’ end of mascRNA. mascRNA is then cleaved by RNase Z and subjected to CCA addition to generate the mature 61-nt mascRNA transcript, which is subsequently exported to the cytoplasm.